Oxysterol-therapeutic agent derivative for bone healing

ABSTRACT

Oxysterol-therapeutic agent derivatives or OXY133-therapeutic agent derivative compounds and methods of synthesizing the same are provided for use in promoting osteogenesis, osteoinduction and/or osteoconduction. Methods of synthesizing in a single container OXY133-therapeutic agent derivatives having high yields and improved process safety are also provided. Methods for synthesizing OXY133-therapeutic agent derivatives that are stereoselective are also provided.

BACKGROUND

Biologics are commonly employed to promote bone growth in medicalapplications including fracture healing and surgical management ofspinal disorders. Spine fusion is often performed by orthopedic surgeonsand neurosurgeons alike to address degenerative disc disease andarthritis affecting the lumbar and cervical spine. Historically,autogenous bone grafting, commonly taken from the iliac crest of thepatient, has been used to augment fusion between vertebral levels.

One protein that is osteoinductive and commonly used to promote spinefusion is recombinant human bone morphogenetic protein-2 (rhBMP-2). Itsuse has been approved by the US Food and Drug Administration (FDA) forsingle-level anterior lumbar interbody fusion. Since this time, the useof rhBMP-2 has increased significantly and expanded to include posteriorlumbar spinal fusion as well as cervical spine fusion.

Oxysterols form a large family of oxygenated derivatives of cholesterolthat are present in the blood, and in human and animal tissues.Oxysterols have been found to be present in atherosclerotic lesions andplay a role in various physiologic processes, such as cellulardifferentiation, inflammation, apoptosis, and steroid production. Somenaturally occurring oxysterols have robust osteogenic properties and canbe used to grow bone. The most potent osteogenic naturally occurringoxysterol, 20(S)-hydroxycholesterol, is both osteogenic andanti-adipogenic when applied to multipotent mesenchymal cells capable ofdifferentiating into osteoblasts and adipocytes.

One such oxysterol is OXY133 or (3S,5S,6S,8R,9S,10R,13S,14S,17S)17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol,which exhibits the following structures:

To synthesize OXY133, often there are complex, multi-step chemicalreactions that are difficult to carry out in a single container. Forexample, to synthesize OXY133 there may be utilization of variousprotection reagents to protect end groups as the molecule is beingsynthesized. In addition, various deprotection reagents are alsoutilized that increase cost, reduce safety and have an adverseenvironmental impact. Further, the route of synthesis of OXY133 can havea very low yield of less than 30%.

Recombinantly produced versions of naturally occurring human proteins,such as rhBMP-2 and rhPDGF, have been studied for decades for theirability to induce or enhance new bone formation. While these proteinshave been effective in supporting bone healing, there are drawbacks withrespect to the complexity of manufacturing and the associated costs. Oneway to address these drawbacks has been to identify small molecules thatregulate parts of the bone signaling pathways to stimulate or enhancebone healing. Examples are the osteoinductive oxysterols and othertherapeutic agents including bisphosphonates, antibiotics, proteins,moieties or fragments thereof.

Therefore, there is a need for a cost effective method of synthesizingoxysterol-therapeutic agent derivatives for use in promotingosteogenesis, osteoinduction and/or osteoconduction. In particular,methods of synthesizing OXY133-therapeutic agent derivatives having ahigh yield and improved process safety that can be scaled-up forindustrial applications would be beneficial. Methods for synthesizing anOXY133-therapeutic agent derivative from endogenous starting material,which is stereoselective, would also be beneficial.

SUMMARY

Oxysterol-therapeutic agent derivatives or OXY133 derivative compoundsand methods of synthesizing the same are provided for use in promotingosteogenesis, osteoinduction and/or osteoconduction. Methods ofsynthesizing in a single container OXY133-therapeutic agent derivativeshaving high yields and improved process safety are also provided.Methods for synthesizing OXY133-therapeutic agent derivatives that arestereoselective are also provided. Methods of synthesizingOXY133-therapeutic agent derivatives that have reduced environmentalimpact and have low product cost are also provided.

In some embodiments, a method of making a derivative of an oxysterol isprovided. The method includes (i) reacting a pregnenolone derivative offormula I: (formula I),

with an organometallic compound to form a diol derivative of formula II:

(ii) subjecting the diol derivative of formula II tohydroboration-oxidation to form a derivative of the oxysterol or apharmaceutically acceptable salt thereof of formula III:

(iii) reacting the compound of formula III with a therapeutic agent or afragment of a therapeutic agent to form an oxysterol derivative offormula IV:

wherein R₁ is a protecting group, for example, methyl, ethyl or silyl,R₃ is an aliphatic or cyclic substituent having at least one carbon, andR4 is a bisphosphonate moiety, an antibiotic moiety or a proteinfragment.

In these embodiments, the organometallic compound is a Grignard reagentcorresponding to the formula R₃MgX, or R₃Li, wherein X is a halide andR₃ is an aliphatic or cyclic substituent having at least one carbon.

In other aspects, the compound of formula IV can be deprotected of R₁ toobtain a compound of formula IVb:

Deprotection can be accomplished by treatment with iodine, a fluoridesource or other suitable deprotection methods.

In some embodiments, the pregnenolone derivative of formula II isprepared by reacting pregnenolone with R₁X in a base, wherein R₁ ismethyl, ethyl, silyl or carbamate, X is a halide and the base is NaOH,KOH or Ca(OH)₂.

In other embodiments, the pregnenolone derivative of formula I isreacted with n-hexyl magnesium chloride in tetrahydrofuran to obtain adiol derivative of the formula IIa:

In other aspects, a compound of formula IIa is subjected tohydroboration-oxidation wherein the borane compound is BH₃ to form aborane intermediate, which is reacted with hydrogen in a base, as forexample, NaOH, KOH or Ca(OH)₂ to form an OXY133 derivative of formulaIIIa:

OH (formula IIIa), wherein C3 is protected by R₁, a protecting group.

In another aspect, the compound of formula IIIa can be reacted with atherapeutic agent R₄, wherein R₄ is a bisphosphonate moiety, anantibiotic moiety, or a protein fragment to form a compound of formulaIVa:

In various aspects, the compound of formula IVa can be optionallydeprotected to obtain a compound of formula IVc:

In various embodiments, the pregnenolone derivative of formula IIa isprepared by reacting pregnenolone with R₁X in a base, wherein R₁ ismethyl, ethyl, silyl or carbamate, X is a halide and the base is NaOH,KOH or Ca(OH)₂.

In yet other embodiments, the compound of formula IIIa can be reactedwith a compound comprising a linker L. Useful linkers L compriseaspartate based linkers, succinate based linkers or urethane basedlinkers. The resulting linker containing intermediate is coupled with amoiety or fragment of a therapeutic agent R₄ to obtain a compound offormula VIII:

wherein R₁ is as defined above and R₄ can be a bisphosphonate moiety, anantibiotic moiety, a protein fragment. In some embodiments, R₄ can bekanamycin or kanamycin fragment.

The compound of formula VIII can be optionally deprotected of R₁ with aniodine source, a fluoride source or other suitable deprotection methodsto obtain a compound of formula VIIIa:

In other aspects, when L is a succinate based linker the oxysterolderivative is a compound of the formula IXa:

In other embodiments, the compound of formula IIIa can be reacted withsuccinic anhydride to provide a succinate based linker and then coupledwith a compound of formula V:

to obtain a compound of formula VIb:

In some embodiments, the compound of formula VIb can be optionallydeprotected of R₁ by treatment with an iodine source or a fluoridesource or other suitable deprotection method to obtain a compound offormula VI (OXY 149):

In other embodiments, this application provides a compound correspondingto the structure of formula X:

wherein R₁ is methyl, ethyl, or silyl, R₃ is (C₆-C₂₆) alkyl orheteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl orheteroalkyl and a (C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, a(C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a (C₄-C₁₀) alkyleno orheteroalkyleno or carbamate or benzyl or silyl, L is a linker moiety,the linker moiety comprising an aspartate based linker, a succinatebased linker or a urethane based linker and R₄ is a therapeutic agent.In other aspects, the therapeutic agent can be a bisphosphonate moiety,an antibiotic moiety or a protein fragment.

In various embodiments, the aspartate based linker, the succinate basedlinker or the urethane based linker have the following formulas:

In various embodiments, the above methods of preparing anoxysterol-therapeutic agent derivative with or without a linker L can becarried out in a single container.

In certain embodiments, this disclosure provides for pharmaceuticalcompositions comprising a compound of Formula X and a pharmaceuticallyacceptable carrier or diluent.

In other embodiments, this disclosure provides a method of treating amammal suffering from a bone disorder, the method comprisingadministering to the mammal an effective amount of theoxysterol-derivative of Formula X, wherein the complex is administeredto the mammal by localized or systemic delivery to the mammal. The bonedisorder comprises a bone fracture, osteoporosis or osteopenia.

In other aspects, this disclosure provides a method of treating a mammalsuffering from a bone disorder, the method comprising administering tothe mammal an effective amount of the compound of Formula X.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIG. 1 illustrates a step-wise reaction for synthesizing OXY133 withstarting reactants comprising pregnenolone acetate, as shown in oneembodiment of this disclosure. The pregnenolone is reacted with anorganometallic compound to produce a sterol or diol having two hydroxylgroups. The sterol or diol is then reacted with borane and hydrogenperoxide and purified to produce OXY133;

FIG. 2 is a graphic illustration of the ¹H NMR data obtained fromisolated and purified OXY133;

FIG. 3 is a graphic illustration of the ¹³C NMR data obtained fromOXY133;

FIG. 4 is a graphic illustration of the infrared spectroscopy dataobtained from OXY133;

FIG. 5 is a graphic illustration of the mass spectroscopy data obtainedfrom OXY133;

FIG. 6 is a graphic illustration of ¹H NMR data obtained from theintermediary sterol or diol to synthesize OXY133;

FIG. 7 is a graphic illustration of ¹³C NMR data obtained from theintermediary sterol or diol to synthesize OXY133;

FIG. 8 is a schematic of a synthesis of oxysterol analogs according toone embodiment of this application;

FIG. 9 is a schematic of a synthesis of oxysterol analogs according toanother embodiment of this application wherein at C20 there is a hexylside chain;

FIG. 10 is a schematic of a synthesis of OXY133 therapeutic analogsaccording to an embodiment of this application;

FIG. 11 is a schematic of a synthesis of OXY149 according to anembodiment of this application; and

FIG. 12 is a schematic of a synthesis of OXY149 methyl ether accordingto another embodiment of this application.

FIG. 13 is a schematic of an oxysterol derivative synthesis, wherein theoxysterol is attached to an alendronate moiety.

FIG. 14 is a schematic of an oxysterol derivative synthesis, wherein theoxysterol is attached to a protein fragment. An example of a proteinfragment is BMP-2.

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present application. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the present application are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements. Moreover, all ranges disclosedherein are to be understood to encompass any and all sub ranges subsumedtherein. For example, a range of “1 to 10” includes any and all subranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all sub ranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

Definitions

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “an alkanolamine” includes one, two, three or morealkanolamines.

The term “bioactive agent” as used herein is generally meant to refer toany substance that alters the physiology of a patient. The term“bioactive agent” may be used interchangeably herein with the terms“therapeutic agent,” “therapeutically effective amount,” and “activepharmaceutical ingredient”, “API” or “drug”. The terms “bioactive”composition or “pharmaceutical” composition are used interchangeablyherein. Both terms refer to compositions that can be administered to asubject, used to coat or are present in a medical device that isintroduced into a subject, or the like.

A “subject,” as used herein, includes any animal that exhibits a symptomof a condition that can be treated with a compound. Suitable subjects(patients) include laboratory animals (such as mouse, rat, rabbit, orguinea pig), farm animals, and domestic animals or pets (such as a cat,dog, or horse). Non-human primates and humans, including human patients,are included. Typical subjects include animals that exhibit aberrantamounts (lower amounts than a “normal” or “healthy” subject) of one ormore physiological activities that are stimulated by Hedgehog signaling.The aberrant activities may be regulated by any of a variety ofmechanisms, including activation of a Hedgehog activity. The aberrantactivities can result in a pathological condition.

The term “biodegradable” includes compounds or components that willdegrade over time by the action of enzymes, by hydrolytic action and/orby other similar mechanisms in the human body. In various embodiments,“biodegradable” includes components that can break down or degradewithin the body to non-toxic components as cells (e.g., bone cells)infiltrate the components and allow repair of the defect. By“bioerodible” it is meant that the compounds or components will erode ordegrade over time due, at least in part, when come into contact withsubstances found in the surrounding tissue, fluids or by cellularaction. By “bioabsorbable” it is meant that the compounds or componentswill be broken down and absorbed within the human body, for example, bya cell or tissue. “Biocompatible” means that the compounds or componentswill not cause substantial tissue irritation or necrosis at the targettissue site and/or will not be carcinogenic.

The term “alkyl” as used herein, refers to a saturated or unsaturated,branched, straight-chain or cyclic monovalent hydrocarbon radicalderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkane, alkene or alkyne. Typical alkyl groups include, but arenot limited to, methyl; ethyl's such as ethanol, ethyl; propyls such aspropan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl,prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkenyl”and/or “alkynyl” is used, as defined below. In some embodiments, thealkyl groups are (C1-C40) alkyl. In some embodiments, the alkyl groupsare (C1-C6) alkyl.

The term “alkenyl” as used herein refers to a saturated branched,straight-chain or cyclic alkyl radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane. Typicalalkenyl groups include, but are not limited to, methanol; ethanol;propanol's such as propan-1-yl, propan-2-yl(isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like. In some embodiments, thealkanyl groups are (C1-C40) alkanyl. In some embodiments, the alkanylgroups are (C1-C6) alkanyl.

The term “alkenyl” as used herein refers to an unsaturated branched,straight-chain or cyclic alkyl radical having at least one carbon-carbondouble bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkene. The radical may be in either the cis ortrans conformation about the double bond(s). Typical alkenyl groupsinclude, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In some embodiments, the alkenyl group is (C2-C40)alkenyl. In some embodiments, the alkenyl group is (C2-C6) alkenyl.

The term “alkynyl” as used herein refers to an unsaturated branched,straight-chain or cyclic alkyl radical having at least one carbon-carbontriple bond derived by the removal of one hydrogen atom from a singlecarbon atom of a parent alkyne. Typical alkynyl groups include, but arenot limited to, ethynyl; propynyls such as prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl, but-3-yn-1-yl,etc.; and the like. In some embodiments, the alkynyl group is (C2-C40)alkynyl. In some embodiments, the alkynyl group is (C2-C6) alkynyl.

The term “alkyldiyl” as used herein refers to a saturated orunsaturated, branched, straight-chain or cyclic divalent hydrocarbonradical derived by the removal of one hydrogen atom from each of twodifferent carbon atoms of a parent alkane, alkene or alkyne, or by theremoval of two hydrogen atoms from a single carbon atom of a parentalkane, alkene or alkyne. The two monovalent radical centers or eachvalence of the divalent radical center can form bonds with the same ordifferent atoms. Typical alkyldiyls include, but are not limited tomethandiyl; ethyldiyls such as ethan-1,1-diyl, ethan-1,2-diyl,ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as propan-1,1-diyl,propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropan-1,1-diyl,cyclopropan-1,2-diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl,prop-2-en-1,2-diyl, prop-1-en-1,3-diyl cycloprop-1-en-1,2-diyl,cycloprop-2-en-1,2-diyl, cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl,etc.; butyldiyls such as, butan-1,1-diyl, butan-1,2-diyl,butan-1,3-diyl, butan-1,4-diyl, butan-2,2-diyl,2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl, cyclobutan-1,1-diyl;cyclobutan-1,2-diyl, cyclobutan-1,3-diyl, but-1-en-1,1-diyl,but-1-en-1,2-diyl, but-1-en-1,3-diyl, but-1-en-1,4-diyl,2-methyl-prop-1-en-1,1-diyl, 2-methanylidene-propan-1,1-diyl,buta-1,3-dien-1,1-diyl, buta-1,3-dien-1,3-diyl, cyclobut-1-en-1,2-diyl,cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Insome embodiments, the alkyldiyl group is (C1-C40) alkyldiyl. In someembodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also contemplatedare saturated acyclic alkanyldiyl radicals in which the radical centersare at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl(ethano); propan-1,3-diyl(propano);butan-1,4-diyl(butano); and the like (also referred to as alkylenos,defined infra).

The term “alkyleno” as used herein refers to a straight-chain alkyldiylradical having two terminal monovalent radical centers derived by theremoval of one hydrogen atom from each of the two terminal carbon atomsof straight-chain parent alkane, alkene or alkyne. Typical alkylenogroups include, but are not limited to, methano; ethylenos such asethano, etheno, ethyno; propylenos such as propano, prop[1]eno,propa[1,2]dieno, prop[1]yno, etc.; butylenos such as butano, but[1]eno,but[2]eno, buta[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno, etc.;and the like. Where specific levels of saturation are intended, thenomenclature alkano, alkeno and/or alkyno is used. In some embodiments,the alkyleno group is (C1-C40) alkyleno. In some embodiments, thealkyleno group is (C1-C6) alkyleno.

The terms “heteroalkyl,” “heteroalkanyl,” “heteroalkenyl,”“heteroalkynyl,” “heteroalkyldiyl” and “heteroalkyleno” as used hereinrefer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkylenoradicals, respectively, in which one or more of the carbon atoms areeach independently replaced with the same or different heteroatomicgroups. Typical heteroatomic groups which can be included in theseradicals include, but are not limited to, —O—, —S—, —O—O—, —S—S—, —O—S—,—NR′, ═N—N═, —N═N—, —N(O)N—, —N═N—NR′—, —PH—, —P(O)2-, —O—P(O)2-, —SH2-,—S(O)2-, or the like, where each R′ is independently hydrogen, alkyl,alkanyl, alkenyl, alkynyl, aryl, arylaryl, arylalkyl, heteroaryl,heteroarylalkyl or heteroaryl-heteroaryl as defined herein.

The term “aryl” as used herein refers to a monovalent aromatichydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, radicals derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like. In some embodiments, the aryl group is (C5-C14) aryl or a(C5-C10) aryl. In some embodiments, the aryls are phenyl and naphthyl.

The term “aryldiyl” as used herein refers to a divalent aromatichydrocarbon radical derived by the removal of one hydrogen atom fromeach of two different carbon atoms of a parent aromatic ring system orby the removal of two hydrogen atoms from a single carbon atom of aparent aromatic ring system. The two monovalent radical centers or eachvalence of the divalent center can form bonds with the same or differentatom(s). Typical aryldiyl groups include, but are not limited to,divalent radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorine, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In someembodiments, the aryldiyl group is (C5-C14) aryldiyl or (C5-C10)aryldiyl. For example, some aryldiyl groups are divalent radicalsderived from benzene and naphthalene, especially phena-1,4-diyl,naphtha-2,6-diyl and naphtha-2,7-diyl.

The term “aryleno” as used herein refers to a divalent bridge radicalhaving two adjacent monovalent radical centers derived by the removal ofone hydrogen atom from each of two adjacent carbon atoms of a parentaromatic ring system. Attaching an aryleno bridge radical, e.g. benzeno,to a parent aromatic ring system, e.g. benzene, results in a fusedaromatic ring system, e.g. naphthalene. The bridge is assumed to havethe maximum number of non-cumulative double bonds consistent with itsattachment to the resultant fused ring system. In order to avoiddouble-counting carbon atoms, when an aryleno substituent is formed bytaking together two adjacent substituents on a structure that includesalternative substituents, the carbon atoms of the aryleno bridge replacethe bridging carbon atoms of the structure. As an example, consider thefollowing structure:

wherein R¹, when taken alone is hydrogen, or when taken together with R²is (C5-C14) aryleno; and R², when taken alone is hydrogen, or when takentogether with R¹ is (C5-C14) aryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is C6 aryleno (benzeno), the resultantcompound is naphthalene. When R¹ taken together with R² is C10 aryleno(naphthaleno), the resultant compound is anthracene or phenanthrene.Typical aryleno groups include, but are not limited to, aceanthryleno,acenaphthyleno, acephenanthtyleno, anthraceno, azuleno, benzeno (benzo),chryseno, coroneno, fluorantheno, fluoreno, hexaceno, hexapheno,hexyleno, as-indaceno, s-indaceno, indeno, naphthalene (naphtho),octaceno, octapheno, octaleno, ovaleno, penta-2,4-dieno, pentaceno,pentaleno, pentapheno, peryleno, phenaleno, phenanthreno, piceno,pleiadeno, pyreno, pyranthreno, rubiceno, triphenyleno, trinaphthaleno,and the like. Where a specific connectivity is intended, the involvedbridging carbon atoms (of the aryleno bridge) are denoted in brackets,e.g., [1,2]benzeno ([1,2]benzo), [1,2]naphthaleno, [2,3]naphthaleno,etc. Thus, in the above example, when R¹ taken together with R² is[2,3]naphthaleno, the resultant compound is anthracene. When R¹ takentogether with R² is [1,2]naphthaleno, the resultant compound isphenanthrene. In one embodiment, the aryleno group is (C5-C14) or(C5-C10).

The term “arylaryl” as used herein refers to a monovalent hydrocarbonradical derived by the removal of one hydrogen atom from a single carbonatom of a ring system in which two or more identical or non-identicalparent aromatic ring systems are joined directly together by a singlebond, where the number of such direct ring junctions is one less thanthe number of parent aromatic ring systems involved. Typical arylarylgroups include, but are not limited to, biphenyl, triphenyl,phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. When thenumber of carbon atoms comprising an arylaryl group is specified, thenumbers refer to the carbon atoms comprising each parent aromatic ring.For example, (C1-C14) arylaryl is an arylaryl group in which eacharomatic ring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl,binaphthyl, phenylnaphthyl, etc. In some instances, each parent aromaticring system of an arylaryl group is independently a (C5-C14) aromatic ora (C1-C10) aromatic. In some embodiments, the arylaryl groups are groupsin which all of the parent aromatic ring systems are identical, e.g.,biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

The term “biaryl” as used herein refers to an arylaryl radical havingtwo identical parent aromatic systems joined directly together by asingle bond. Typical biaryl groups include, but are not limited to,biphenyl, binaphthyl, bianthracyl, and the like. In some instances, thearomatic ring systems are (C5-C14) aromatic rings or (C5-C10) aromaticrings. In one embodiment, the biaryl group is biphenyl.

The term “arylalkyl” as used herein refers to an acyclic alkyl radicalin which one of the hydrogen atoms bonded to a carbon atom, typically aterminal or sp2 carbon atom, is replaced with an aryl radical. Typicalarylalkyl groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In some embodiments, the arylalkyl group is(C6-C40) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C26) and the aryl moiety is (C5-C14). In someembodiments, the arylalkyl group is (C6-C13), e.g., the alkanyl, alkenylor alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moietyis (C5-C10).

The term “heteroaryl” as used herein refers to a monovalentheteroaromatic radical derived by the removal of one hydrogen atom froma single atom of a parent heteroaromatic ring system. Typical heteroarylgroups include, but are not limited to, radicals derived from acridine,arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. In some embodiments, the heteroaryl group is a 5-14 memberedheteroaryl or a 5-10 membered heteroaryl. In some embodiments, theheteroaryl radicals are those derived from parent heteroaromatic ringsystems in which any ring heteroatoms are nitrogens, such as imidazole,indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

The term “heteroaryldiyl” refers to a divalent heteroaromatic radicalderived by the removal of one hydrogen atom from each of two differentatoms of a parent heteroaromatic ring system or by the removal of twohydrogen atoms from a single atom of a parent heteroaromatic ringsystem. The two monovalent radical centers or each valence of the singledivalent center can form bonds with the same or different atom(s).Typical heteroaryldiyl groups include, but are not limited to, divalentradicals derived from acridine, arsindole, carbazole, β-carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In some embodiments, theheteroaryldiyl group is 5-14 membered heteroaryldiyl or a 5-10 memberedheteroaryldiyl. In some embodiments, heteroaryldiyl groups are divalentradicals derived from parent heteroaromatic ring systems in which anyring heteroatoms are nitrogens, such as imidazole, indole, indazole,isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine,pyrazole, pyrazine, pyridazine, pyridine, pyrrole, quinazoline,quinoline, etc.

The term “heteroaryleno” as used herein refers to a divalent bridgeradical having two adjacent monovalent radical centers derived by theremoval of one hydrogen atom from each of two adjacent atoms of a parentheteroaromatic ring system. Attaching a heteroaryleno bridge radical,e.g. pyridino, to a parent aromatic ring system, e.g., benzene, resultsin a fused heteroaromatic ring system, e.g., quinoline. The bridge isassumed to have the maximum number of non-cumulative double bondsconsistent with its attachment to the resultant fused ring system. Inorder to avoid double-counting ring atoms, when a heteroarylenosubstituent is formed by taking together two adjacent substituents on astructure that includes alternative substituents, the ring atoms of theheteroaryleno bridge replace the bridging ring atoms of the structure.As an example, consider the following structure:

wherein R¹, when taken alone is hydrogen, or when taken together with R²is 5-14 membered heteroaryleno; and R², when taken alone is hydrogen, orwhen taken together with R¹ is 5-14 membered heteroaryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is a 6-membered heteroaryleno pyridino,the resultant compound is isoquinoline, quinoline or quinolizine. WhenR¹ taken together with R² is a 10-membered heteroaryleno (e.g.,isoquinoline), the resultant compound is, e.g., acridine orphenanthridine. Typical heteroaryleno groups include, but are notlimited to, acridino, carbazolo, β-carbolino, chromeno, cinnolino,furan, imidazolo, indazoleno, indoleno, indolizino, isobenzofurano,isochromeno, isoindoleno, isoquinolino, isothiazoleno, isoxazoleno,naphthyridino, oxadiazoleno, oxazoleno, perimidino, phenanthridino,phenanthrolino, phenazino, phthalazino, pteridino, purino, pyrano,pyrazino, pyrazoleno, pyridazino, pyridino, pyrimidino, pyrroleno,pyrrolizino, quinazolino, quinolino, quinolizino, quinoxalino,tetrazoleno, thiadiazoleno, thiazoleno, thiopheno, triazoleno, xantheno,or the like. Where a specific connectivity is intended, the involvedbridging atoms (of the heteroaryleno bridge) are denoted in brackets,e.g., [1,2]pyridino, [2,3]pyridino, [3,4]pyridino, etc. Thus, in theabove example, when R¹ taken together with R² is [1,2]pyridino, theresultant compound is quinolizine. When R¹ taken together with R2 is[2,3]pyridino, the resultant compound is quinoline. When R¹ takentogether with R² is [3,4]pyridino, the resultant compound isisoquinoline. In some embodiments, the heteroaryleno group is 5-14membered heteroaryleno or 5-10 membered heteroaryleno. In someembodiments, the heteroaryleno radicals are those derived from parentheteroaromatic ring systems in which any ring heteroatoms are nitrogens,such as imidazolo, indolo, indazolo, isoindolo, naphthyridino,pteridino, isoquinolino, phthalazino, purino, pyrazolo, pyrazino,pyridazino, pyndmo, pyrrolo, quinazolino, quinolino, etc.

The term “heteroaryl-heteroaryl” as used herein refers to a monovalentheteroaromatic radical derived by the removal of one hydrogen atom froma single atom of a ring system in which two or more identical ornon-identical parent heteroaromatic ring systems are joined directlytogether by a single bond, where the number of such direct ringjunctions is one less than the number of parent heteroaromatic ringsystems involved. Typical heteroaryl-heteroaryl groups include, but arenot limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc.When the number of ring atoms are specified, the numbers refer to thenumber of atoms comprising each parent heteroaromatic ring system. Forexample, 5-14 membered heteroaryl-heteroaryl is a heteroaryl-heteroarylgroup in which each parent heteroaromatic ring system comprises from 5to 14 atoms, e.g., bipyridyl, tripyridyl, etc. In some embodiments, eachparent heteroaromatic ring system is independently a 5-14 memberedheteroaromatic, or a 5-10 membered heteroaromatic. In some embodiments,heteroaryl-heteroaryl groups are groups in which all of the parentheteroaromatic ring systems are identical. In some embodiments,heteroaryl-heteroaryl radicals are those in which each heteroaryl groupis derived from parent heteroaromatic ring systems in which any ringheteroatoms are nitrogens, such as imidazole, indole, indazole,isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine,pyrazole, pyrazine, pyridazine, pyridine, pyrrole, quinazoline,quinoline, etc.

The term “biheteroaryl” as used herein refers to a heteroaryl-heteroarylradical having two identical parent heteroaromatic ring systems joineddirectly together by a single bond. Typical biheteroaryl groups include,but are not limited to, bipyridyl, bipurinyl, biquinolinyl, and thelike. In some embodiments, the heteroaromatic ring systems are 5-14membered heteroaromatic rings or 5-10 membered heteroaromatic rings. Insome embodiments, biheteroaryl radicals are those in which theheteroaryl groups are derived from a parent heteroaromatic ring systemin which any ring heteroatoms are nitrogens, such as biimidazolyl,biindolyl, biindazolyl, biisoindolyl, binaphthyridinyl, bipteridinyl,biisoquinolinyl, biphthalazinyl, bipurinyl, bipyrazolyl, bipyrazinyl,bipyridazinyl, bipyridinyl, bipyrrolyl, biquinazolinyl, biquinolinyl,etc.

The term “heteroarylalkyl” as used herein refers to an acyclic alkylradical in which one of the hydrogen atoms bonded to a carbon atom,typically a terminal or sp2 carbon atom, is replaced with a heteroarylradical. Where specific alkyl moieties are intended, the nomenclatureheteroarylalkanyl, heteroarylakenyl and/or heterorylalkynyl is used. Insome embodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a 5-14membered heteroaryl. In some embodiments, the heteroarylalkyl is a 6-13membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moietyis 1-3 membered and the heteroaryl moiety is a 5-10 membered heteroaryl.

The term “substituted” as used herein refers to a radical in which oneor more hydrogen atoms are each independently replaced with the same ordifferent substituent(s). Typical substituents include, but are notlimited to, —X, —R, —O—, ═O, —OR, —O—OR, —SR, —S—, ═S, —NRR, ═NR,perhalo (C1-C6) alkyl, —CX3, —CF3, —CN, —OCN, —SCN, —NCO, —NCS, —NO,—NO2, ═N2, —N3, —S(O)2O—, —S(O)2OH, —S(O)2R, —C(O)R, —C(O)X, —C(S)R,—C(S)X, —C(O)OR, —C(O)O—, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRRand —C(NR)NRR, where each X is independently a halogen (e.g., —F or —Cl)and each R is independently hydrogen, alkyl, alkanyl, alkenyl, alkanyl,aryl, arylalkyl, arylaryl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl, as defined herein. The actual substituentsubstituting any particular group will depend upon the identity of thegroup being substituted.

The term “solvate” as used herein refers to an aggregate that comprisesone or more molecules of a compound of the disclosure with one or moremolecules of solvent. Examples of solvents that form solvates include,but are not limited to, water, isopropanol, ethanol, methanol, DMSO,ethyl acetate, acetic acid, and ethanolamine. The term “hydrate” refersto the aggregate or complex where the solvent molecule is water. Thesolvent may be inorganic solvents such as, for example, water in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent, such as ethanol. Thus, the compounds of the presentdisclosure may exist as a hydrate, including a monohydrate, dihydrate,hemihydrate, sesquihydrate, trihydrate, tetrahydrate or the like, aswell as the corresponding solvated forms. The compound of the disclosuremay be true solvates, while in other cases, the compound of thedisclosure may merely retain adventitious water or be a mixture of waterplus some adventitious solvent.

The term “oxysterol” as used herein is meant to encompass one or moreforms of oxidized cholesterol. The oxysterols described herein areeither independently or collectively active to bone growth in a patient,as described in WO 2013169399 A1, which is hereby incorporated byreference in its entirety.

The oxysterol, sterol or diol can be in a pharmaceutically acceptablesalt. Some examples of potentially pharmaceutically acceptable saltsinclude those salt-forming acids and bases that do not substantiallyincrease the toxicity of a compound, such as, salts of alkali metalssuch as magnesium, potassium and ammonium, salts of mineral acids suchas hydrochloride, hydriodic, hydrobromic, phosphoric, metaphosphoric,nitric and sulfuric acids, as well as salts of organic acids such astartaric, acetic, citric, malic, benzoic, glycollic, gluconic, gulonic,succinic, arylsulfonic, e.g., p-toluenesulfonic acids, or the like.

Pharmaceutically acceptable salts of oxysterol, sterol or diol includesalts prepared from pharmaceutically acceptable non-toxic bases or acidsincluding inorganic or organic bases, inorganic or organic acids andfatty acids. Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethyl amine, tripropylamine,tromethamine, and the like. When the compound of the current applicationis basic, salts may be prepared from pharmaceutically acceptablenon-toxic acids, including inorganic and organic acids. Such acidsinclude acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Fatty acid salts may also beused, e.g., fatty acid salts having greater than 2 carbons, greater than8 carbons or greater than 16 carbons, such as butyric, caproic,caprylic, capric, lauric, mystiric, palmitic, stearic, arachidic or thelike.

In some embodiments, in order to reduce the solubility of the oxysterol,sterol, or diol to assist in obtaining a controlled release depoteffect, the oxysterol, sterol, or diol is utilized as the free base orutilized in a salt which has relatively lower solubility. For example,the present application can utilize an insoluble salt such as a fattyacid salt. Representative fatty acid salts include salts of oleic acid,linoleic acid, or fatty acid salts with between 8 to 20 carbonssolubility, such as for example, palmeate or stearate.

The term “solvate” is a complex or aggregate formed by one or moremolecules of a solute, e.g. a compound or a pharmaceutically-acceptablesalt thereof, and one or more molecules of a solvent. Such solvates canbe crystalline solids having a substantially fixed molar ratio of soluteand solvent. Suitable solvents include for example, water, ethanol, etc.

The terms “bioactive” composition or “pharmaceutical” composition asused herein may be used interchangeably. Both terms refer tocompositions that can be administered to a subject. Bioactive orpharmaceutical compositions are sometimes referred to herein as“pharmaceutical compositions” or “bioactive compositions” of the currentdisclosure. Sometimes the phrase “administration of OXY133” is usedherein in the context of administration of this compound to a subject(e.g., contacting the subject with the compound, injecting the compound,administering the compound in a drug depot, etc.). It is to beunderstood that the compound for such a use can generally be in the formof a pharmaceutical composition or bioactive composition comprising theOXY133.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the oxysterol (e.g., OXY133), sterol, diol, resultsin alteration of the biological activity, such as, for example,enhancing bone growth, etc. The dosage administered to a patient can beas single or multiple doses depending upon a variety of factors,including the drug's administered pharmacokinetic properties, the routeof administration, patient conditions and characteristics (sex, age,body weight, health, size, etc.), and extent of symptoms, concurrenttreatments, frequency of treatment and the effect desired. In someembodiments, the formulation is designed for immediate release. In otherembodiments, the formulation is designed for sustained release. In otherembodiments, the formulation comprises one or more immediate releasesurfaces and one or more sustained release surfaces.

A “pharmaceutically acceptable carrier” is meant as a material that isnot biologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.

The terms “analogue” or “derivative” as used herein relate to a chemicalmolecule that is similar to another chemical substance in structure andfunction, often differing structurally by a single element or group,which may differ by modification of more than one group (e.g., 2, 3, or4 groups) if it retains the same function as the parental chemicalsubstance. Such modifications are routine to one of ordinary skill inthe art, and include, for example, additional or substituted chemicalmoieties, such as esters or amides of an acid, protecting groups such asa benzyl group for an alcohol or thiol, and tert-butoxylcarbonyl groupsfor an amine. Also included are modifications to alkyl side chains, suchas alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.),modifications to the level of saturation or unsaturation of side chains,and the addition of modified groups such as substituted phenyl andphenoxy. Derivatives can also include conjugates, such as biotin oravidin moieties, enzymes such as horseradish peroxidase and the like,and radio-labeled, bioluminescent, chemoluminescent, or fluorescentmoieties. Further, moieties can be added to the agents described hereinto alter their pharmacokinetic properties, such as to increase half-lifein vivo or ex vivo, or to increase their cell penetration properties,among other desirable properties. Also included are OXY133 derivatives,which are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing,etc.). The term “derivative” also includes within its scope alterationsthat have been made to a parent sequence including additions, deletions,and/or substitutions that provide for functionally equivalent orfunctionally improved molecules.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of two or more amino acids linked covalently throughpeptide bonds. The protein or polypeptide can be coupled to theoxysterol by, for example, covalent, non-covalent or hydrogen bonding.The terms do not refer to a specific length of the product. Thus,“peptides,” and “oligopeptides,” are included within the definition ofpolypeptide.

A “peptide,” as meant herein, is a polypeptide that comprises a shortamino acid sequence, which may or may not be glycosylated and/or containmodified amino acids. A peptide can be from 2 to 75 amino acids long. Insome embodiments, a peptide is 3-60, 3-50, 3-40, 3-30, or 3-20 aminoacids long. In other embodiments, a peptide can be 5-25, 5-15, 10-20, or20-30 amino acids long. In other embodiments, a peptide can be about, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 amino acids long.

As used herein “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout and refer to a molecule comprising two ormore amino acid residues joined to each other by peptide bonds.Peptides, polypeptides and proteins are also inclusive of modificationsincluding, but not limited to, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation. Polypeptides can be of scientific or commercialinterest, including protein-based drugs. Polypeptides include, amongother things, antibodies, fusion proteins, and cytokines. Peptides,polypeptides and proteins are produced by recombinant animal cell linesusing cell culture methods and may be referred to as “recombinantpeptide”, “recombinant polypeptide” and “recombinant protein”.

The term “protein fragment” refers to a protein in which amino acidresidues are deleted as compared to the reference protein itself, butwhere the remaining amino acid sequence is usually identical to orsubstantially identical (for example, from 100%, 99%, 98%, 97%, 96%,95%, 90%, 85%, 80%, 75%, 70%, 65%, to about 60% identical) to that ofthe reference protein. Such deletions may occur at the amino-terminus orcarboxy-terminus of the reference protein, or alternatively both.Deletions may also occur internally. In some embodiments, the proteinfragment can be less than the entire protein but is similar to theentire protein in structure and function, often differing structurallyby a single element or group, which may differ by modification of morethan one group (e.g., 2, 3, or 4 groups) that is/are removed or changedto accomplish attachment to the oxysterol, however, the protein fragmentretains the same function as the entire protein. A suitable proteinfragment is bone morphogenetic protein-2.

As used herein, “moiety” refers to a chemical molecule that is similarto another chemical molecule in structure and function, often differingstructurally by a single element or group, which may differ bymodification of more than one group (e.g., 2, 3, or 4 groups) if itretains the same function as the parental chemical substance. Forexample, when R4 is an antibiotic or a bisphosphonate, the antibioticmoiety or the bisphosphonate moiety will be attached to the oxysteroland often differ structurally by a single element or group, however, theantibiotic moiety or a bisphosphonate moiety will retain the samefunction as the parental chemical substance.

A “depot” includes but is not limited to capsules, microspheres,microparticles, microcapsules, microfiber particles, nanospheres,nanoparticles, coating, matrices, wafers, pills, pellets, emulsions,liposomes, micelles, gels, or other pharmaceutical delivery compositionsor a combination thereof. In some embodiments, suitable materials forthe depot are pharmaceutically acceptable biodegradable and/or anybioabsorbable materials that are FDA approved or GRAS materials. Thesematerials can be polymeric or non-polymeric, as well as synthetic ornaturally occurring, or a combination thereof.

The term “implantable” as utilized herein refers to a biocompatibledevice (e.g., drug depot) retaining potential for successful placementwithin a mammal. The expression “implantable device” and expressions ofthe like as utilized herein refers to an object implantable throughsurgery, injection, or other suitable means whose primary function isachieved either through its physical presence or mechanical properties.

“Localized” delivery includes delivery where one or more drugs aredeposited within a tissue, for example, a bone cavity, or in closeproximity (within about 0.1 cm, or preferably within about 10 cm, forexample) thereto. For example, the drug dose delivered locally from thedrug depot may be, for example, 1%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, 99.9% or 99.999% less than the oral dosage or injectabledose.

The term “mammal” refers to organisms from the taxonomy class“mammalian” including, but not limited to, humans, other primates suchas chimpanzees, apes, orangutans and monkeys, rats, mice, cats, dogs,cows, horses, etc.

The oxysterol can be “osteogenic,” where it can enhance or acceleratethe ingrowth of new bone tissue by one or more mechanisms such asosteogenesis, osteoconduction and/or osteoinduction.

New compositions and methods are provided for efficiently and safelysynthesize oxysterols and oxysterol derivatives for industrialapplications. In some embodiments, this disclosure provides methods ofmaking OXY133 analogs or derivatives including OXY133 therapeuticanalogs suitable for scale-up or industrial application. Methods andcompositions that can efficiently and safely generate OXY133 therapeuticanalogs or derivatives are also provided.

The section headings below should not be restricted and can beinterchanged with other section headings.

Oxysterols

The present disclosure includes an osteogenic oxysterol (e.g., OXY133),sterol, or diol and its ability to promote osteogenic differentiation invitro. OXY133 is a particularly effective osteogenic agent. In variousapplications, OXY133 is useful in treating conditions that would benefitfrom localized stimulation of bone formation, such as, for example,spinal fusion, fracture repair, bone regenerative/tissue applications,augmentation of bone density in the jaw for dental implants,osteoporosis or the like. One particular advantage of OXY133 is that itprovides greater ease of synthesis and improved time to fusion whencompared to other osteogenic oxysterols. OXY133 is a small molecule thatcan serve as an anabolic therapeutic agent for bone growth, as well as auseful agent for treatment of a variety of other conditions.

One aspect of the application disclosure is a compound, named OXY133,having the formula:

or a pharmaceutically acceptable salt, solvate or hydrate thereof. TheOXY133 may be used as a bioactive or pharmaceutical compositioncomprising OXY133 or a pharmaceutically acceptable salt, solvate orhydrate thereof and a pharmaceutically acceptable carrier.

Another aspect of the disclosure is a method for inducing (stimulating,enhancing) a hedgehog (Hh) pathway mediated response, in a cell ortissue, comprising contacting the cell or tissue with a therapeuticallyeffective amount of OXY133. The cell or tissue can be in vitro or in asubject, such as a mammal. The hedgehog (Hh) pathway mediated responseinvolves the stimulation of osteoblastic differentiation,osteomorphogenesis, and/or osteoproliferation, the stimulation of hairgrowth and/or cartilage formation; the stimulation of neovasculogenesis,e.g. angiogenesis, thereby enhancing blood supply to ischemic tissues;or it is the inhibition of adipocyte differentiation, adipocytemorphogenesis, and/or adipocyte proliferation; or the stimulation ofprogenitor cells to undergo neurogenesis. The Hh mediated response cancomprise the regeneration of any of a variety of types of tissues, foruse in regenerative medicine. Another aspect of the disclosure is amethod for treating a subject having a bone disorder, osteopenia,osteoporosis, or a bone fracture comprising administering to the subjectan effective amount of a bioactive composition or pharmaceuticalcomposition comprising OXY133. The subject can be administered thebioactive composition or pharmaceutical composition at a therapeuticallyeffective dose in an effective dosage form at a selected interval to,e.g., increase bone mass, ameliorate symptoms of osteoporosis, reduce,eliminate, prevent or treat atherosclerotic lesions, or the like. Thesubject can be administered the bioactive composition or pharmaceuticalcomposition at a therapeutically effective dose in an effective dosageform at a selected interval to ameliorate the symptoms of osteoporosis.In some embodiments, a composition comprising OXY133 may includemesenchymal stem cells to induce osteoblastic differentiation of thecells at a targeted surgical area.

In various aspects, the OXY133 can be administered to a cell, tissue ororgan by local administration. For example, the OXY133 can be appliedlocally with a cream or the like, or it can be injected or otherwiseintroduced directly into a cell, tissue or organ, or it can beintroduced with a suitable medical device, such as a drug depot asdiscussed herein.

In some embodiments, the dosage of OXY133, sterol, or diol is fromapproximately 10 pg/day to approximately 80 mg/day. Additional dosagesof OXY133, sterol, or diol include from approximately 2.4 ng/day toapproximately 50 mg/day; approximately 50 ng/day to approximately 2.5mg/day; approximately 250 ng/day to approximately 250 mcg/day;approximately 250 ng/day to approximately 50 mcg/day; approximately 250ng/day to approximately 25 mcg/day; approximately 250 ng/day toapproximately 1 mcg/day; approximately 300 ng/day to approximately 750ng/day or approximately 0.50 mcg/day to 500 ng/day. In variousembodiments, the dose may be about 0.01 to approximately 10 mcg/day orapproximately 1 ng/day to about 120 mcg/day.

In addition to the compound OXY133, sterol, or diol other embodiments ofthe disclosure encompass any and all individual stereoisomers at any ofthe stereocenters present in OXY133, including diastereomers, racemates,enantiomers, and other isomers of the compound. In some embodiments ofthe disclosure, OXY133, sterol, oxysterol, diol may include allpolymorphs, solvates or hydrates of the compound, such as hydrates andthose formed with organic solvents.

The ability to prepare salts depends on the acidity or basicity of acompound. Suitable salts of the compound include, but are not limitedto, acid addition salts, such as those made with hydrochloric,hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as sodium and potassiumsalts; alkaline earth metal salts, such as calcium and magnesium salts;and salts formed with organic or inorganic ligands, such as quaternaryammonium salts. Additional suitable salts include, but are not limitedto, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate,pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate,tosylate, triethiodide and valerate salts of the compounds.

In various embodiments, OXY133, sterol, or diol includes one or morebiological functions. That is, OXY133, sterol, or diol can induce abiological response when contacted with a mesenchymal stem cell or abone marrow stromal cell. For example, OXY133, sterol, or diol maystimulate osteoblastic differentiation. In some embodiments, a bioactivecomposition including OXY133 sterol, or diol may include one or morebiological functions when administered to a mammalian cell, for example,a cell in vitro or a cell in a human or an animal. For example, such abioactive composition may stimulate osteoblastic differentiation. Insome embodiments, such a biological function can arise from stimulationof the hedgehog pathway.

Methods of Making an Intermediary Diol

In some embodiments, the current disclosure provides a method for thepreparation of an intermediary diol used in the production of OXY133, asshown below. The diol may be used to promote bone growth as well.Previous methods of synthesis for OXY133 production were inefficient andnot suitable for scale up manufacturing. Some stereoisomers of OXY133perform less optimally than others. The disclosed method isstereoselective and produces a high yield of the specific isomeric formof the diol shown below, which has been shown to produce an optimallyeffective isomeric form of OXY133.

Disclosed are multiple embodiments of reactions to synthesize theintermediary diol. The diol synthesized has the IUPAC designation(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-H-cyclopenta[a]phenanthren-3-ol.Generally, the method of synthesizing the diol includes reactingpregnenolone, pregnenolone acetate or a pregnenolone derivative with anorganometallic reagent to facilitate alkylation of the C20 position, asshown below:

In one embodiment, as shown above in scheme 1, pregnenolone acetate(Formula 1) may be alkylated by an organometallic reagent to synthesizethe intermediary diol, shown above as Formula 2. In some embodiments,pregnenolone acetate is reacted with a Grignard reagent to facilitatealkylation of the C20 position on the pregnenolone acetate molecule. Insome embodiments, n-hexylmagnesium chloride is used as theorganometallic reagent.

In some embodiments, as shown above as scheme 2, pregnenolone is reactedwith a Grignard reagent such as n-hexylmagnesium chloride to facilitatealkylation of the C20 position of the pregnenolone molecule to form theintermediary diol shown as Formula 2.

The method of synthesizing the intermediary diol (Formula 2) or(3S,8S,9S,1 OR, 13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-olis stereoselective and produces a high yield of the diol. For example,in some embodiments, the yield of the desired stereoisomer of the diolis between about 60% and about 70%. In some embodiments, the yield ofthe desired stereoisomer of the diol is between about 50% and about 60%.However, it is contemplated that the percent yield may be higher orlower than these amounts. For example, the percent yield of Formula 2 asshown above may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or 95%. In some embodiments, the percentyield may be above 95%.

In various embodiments, the alkylation reaction is carried out in apolar organic solvent, such as tetrahydrofuran. However, the reactionmay be carried out in a variety of polar organic solvents. For example,the reaction may be carried out in diethyl ether, ethyl ether, dimethylether or the like.

In some embodiments, pregnenolone or pregnenolone acetate is used as astarting reactant. However, in other embodiments, derivatives ofpregnenolone acetate may be used. For example, other specific examplesof compounds which could be used in the present disclosure include:pregnenolone sulfate, pregnenolone phosphate, pregnenolone formate,pregnenolone hemioxalate, pregnenolone hemimalonate, pregnenolonehemiglutarate, 20-oxopregn-5-en-30-yl carboxymethyl ether,3β-hydroxypregn-5-en-20-one sulfate,3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one,3-hydroxy-19-norpregna-1,3,5(10),6,8-pentaen-20-one, 17α-isopregnenolonesulfate, 17-acetoxypregnenolone sulfate, 21-hydroxypregnenolone sulfate,20β-acetoxy-3β-hydroxypregn-5-ene-sulfate, pregnenolone sulfate20-ethyleneketal, pregnenolone sulfate 20-carboxymethyloxime,20-deoxypregnenolone sulfate, 21-acetoxy-17-hydroxypregnenolone sulfate,17-propyloxypregnenolone sulfate, 17-butyloxypregnenolone sulfate,21-thiol esters of pregnenolone sulfate, pyridinium, imidazolium,6-methylpregnenolone sulfate, 6,16α-dimethylpregnenolone sulfate,3β-hydroxy-6-methylpregna-5,16-dien-20-one sulfate,3β-hydroxy-6,16-dimethylpregna-5,16-dien-20-one sulfate,3-hydroxypregna-5,16-dien-20-one sulfate, diosgenin sulfate,3β-hydroxyandrost-5-en-17β-carboxylic acid methyl ester sulfate,3αhydroxy-5β-pregnan-20-one formate, 3α-hydroxy-5β-pregnan-20-onehemioxalate, 3α-hydroxy-5β-pregnan-20-one hemimalonate,3α-hydroxy-5β-pregnan-20-one hemisuccinate, 3α-hydroxy-5β-pregnan-20-onehemiglutarate, estradiol-3-formate, estradiol-3-hemioxalate,estradiol-3-hemimalonate, estradiol-3-hemisuccinate,estradiol-3-hemiglutarate, estradiol-17-methyl ether,estradiol-17-formate, estradiol-17-hemioxalate,estradiol-17-hemimalonate, estradiol-17-hemisuccinate,estradiol-17-hemiglutarate, estradiol-3-methyl ether, 17-deoxyestrone,and 1713-hydroxyestra-1,3,5(10)-trien-3-yl carboxymethyl ether.

In some embodiments, the organometallic comprises n-hexylmagnesiumchloride. However, in some embodiments, the alkylation reaction may becarried out with the use of an alkyllithium, such as, for example,n-hexyllithium. In various embodiments, the organometallic includes analkyl halide. For example, the organometallic reagent may have thefollowing formula:

R—Mg—X,

where Mg comprises magnesium, X comprises chlorine, bromine, fluorine,iodine, or astatine and R comprises an alkyl, a heteroalkyl, an alkanyl,a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkynyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an aryleno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R substituent comprises a (C1-C20)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R substituent is an aliphatic group. In someembodiments, the R substituent is a cyclic group. In some embodiments,the R substituent is a hexyl group.

Alternatively, the organometallic may comprise the formula:

R—Li,

where Li comprises lithium and R comprises an alkyl, a heteroalkyl, analkanyl, a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkynyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an aryleno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R substituent comprises a (C₁-C₂₀)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R substituent is an aliphatic group. In someembodiments, the R substituent is a cyclic group. In some embodiments,the R substituent is a hexyl group.

In some embodiments, the alkylation reaction is exothermic and thereaction vessel may be temperature controlled to maintain optimalreaction kinetics. In some embodiments, the exothermic reaction releasesabout 1000 BTU per pound of solution. Due to the strongly exothermicnature of the reaction, the Grignard reagent therefore must be addedslowly so that volatile components, for example ethers, are notvaporized due to the reaction heat. In some embodiments, the reactionvessel may be cooled by internal cooling coils. The cooling coils may besupplied with a coolant by means of an external gas/liquid refrigerationunit. In some embodiments, an internal temperature of the reactionvessel is maintained at less than 15° C., 10° C., 5° C. or 1° C. In someembodiments, the reaction vessel is maintained at about 0° C. during thealkylation reaction to form the intermediary diol of Formula 2.

In various embodiments, the diol of Formula 2 is synthesized along withbyproducts and can be purified. For example, the resulting diol ofFormula 2 may be a byproduct of a diastereomeric mixture. In variousembodiments, the diol of Formula 2 may be isolated and purified. Thatis, the diol of formula 2 can be isolated and purified to the desiredpurity, e.g., from about 95% to about 99.9% by filtration,centrifugation, distillation, which separates volatile liquids on thebasis of their relative volatilities, crystallization,recrystallization, evaporation to remove volatile liquids fromnon-volatile solutes, solvent extraction to remove impurities,dissolving the composition in a solvent in which other components aresoluble therein or other purification methods. The diol may be purifiedby contacting it with organic and/or inorganic solvents, for example,THF, water, diethyl ether, dichloromethane, ethyl acetate, acetone,n,n-dimethylformamide, acetonitrile, dimethyl sulfoxide, ammonia,t-butanol, n-propanol, ethanol, methanol, acetic acid, or a combinationthereof.

In various embodiments, the alkylation step and the purification steptake place in the same reaction vessel.

In some embodiments, the diol is quenched with aqueous ammonium chlorideor acetic acid to reduce the amount of anions present and neutralize thereaction which is then separated from the resulting organic layer. Theseparated residue is recovered by evaporation and purified by silica gelcolumn chromatography.

The diol may be anhydrous or in the monohydrate form. However, in otherembodiments the purified diol may be crystallized in other hydrousforms, such as, for example, a dihydrate, a hemihydrate, asesquihydrate, a trihydrate, a tetrahydrate and the like, as well as thecorresponding solvated forms. In other embodiments, the purified diol iscrystallized as a co-crystal or a pharmaceutically acceptable salt.

Methods of Making OXY133

In some embodiments, the current disclosure provides a method for thepreparation of an OXY133, as shown below. Previous methods of synthesisfor OXY133 produce diastereomeric mixtures of OXY133 intermediates whichrequire purification methods to separate. As discussed above to form theintermediary diol, the disclosed method is stereoselective and producesa high yield of the specific isomeric forms of OXY133. The formula ofOXY133 is shown below.

Disclosed are multiple embodiments of reactions to synthesize OXY133.OXY133 has the IUPAC designation (3S,5S,6S,8R,9S,1OR,13S,14S,17S)-17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol.OXY133 has previously been synthesized through a complex process notsuitable for scale-up as shown below:

However, the reaction has difficulty being carried out in a singlecontainer. The reaction shown above involves more reagents to carry outreaction steps (e.g., blocking and deprotection groups and steps) whichhave an adverse environmental impact. Additionally, the known methodsinvolve reagents that are expensive and often difficult to obtain.Further, the method shown in Scheme 3 gives relatively low yields, hasmore degradation products, impurities and creates many toxic byproducts.

Generally, the method of synthesizing OXY133 as disclosed hereinincludes reacting the diol synthesized as described herein with boranein the reaction shown below:

In some embodiments, crude and unpurified OXY133 is produced through ahydroboration and oxidation reaction of the intermediary diol havingFormula 2 in reaction Scheme 4. Borane compounds that can be used in thereaction include BH₃, B₂H₆, BH₃S(CH₃)₂ (BMS), borane adducts withphosphines and amines, e.g., borane triethylamine; monosubstitutedboranes of the form RBH₂ where R=alkyl and halide, monoalkyl boranes(e.g., IpcBH2, monoisopinocampheylborane), monobromo- andmonochloro-borane, complexes of monochloroborane and 1,4-dioxane,disubstituted boranes including bulky boranes, such as for example,dialkylborane compounds such as diethylborane,bis-3-methyl-2-butylborane (disiamylborane), 9-borabycyclo[3,3,1]nonane(9-BBN), disiamylborane (Sia₂BH), dicyclohexylborane (Chx2BH),trialkylboranes, dialkylhalogenoboranes, dimesitylborane (C₆H₂Me₃)₂BH,alkenylboranes, pinacolborane, or catecholborane or a combinationthereof.

Briefly, a hydroboration and oxidation reaction is a two-step reaction.The boron and hydrogen add across the double bond of an alkene to form acomplex with the alkene. Thus the boration phase of the reaction isstereoselective and regioselective. The oxidation phase of the reactioninvolves basic aqueous hydrogen peroxide to furnish a hydroxylsubstituent in place of the boron. See Vollhart, K P, Schore, N E, 2007,Organic Chemistry: Structure and Function, Fifth Ed., New York, N.Y.,Custom Publishing Company. Thus, the intermediary diol having Formula 2is reacted with borane and hydrogen peroxide to form crude OXY133. Insome embodiments, the step of forming crude OXY133 takes place in thesame reaction vessel as the alkylation reaction. In other embodiments,the step of forming crude OXY133 takes place in a different reactionvessel as the alkylation reaction.

The hydroboration-oxidation step of the synthesis of OXY133, like thestep of forming the intermediary diol, is stereoselective and produces ahigh yield. For example, in some embodiments, the percent yield of crudeOXY133 may be higher or lower than these amounts. For example, thepercent yield of Formula 2 as shown above may be about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Insome embodiments, the percent yield may be above 95%.

In various embodiments, the hydroboration-oxidation reaction is carriedout in a polar organic solvent, such as tetrahydrofuran. However, thereaction may be carried out in a variety of polar organic solvents. Forexample, the reaction may be carried out in diethyl ether, ethyl ether,dimethyl ether or the like.

In some embodiments, the hydroboration-oxidation reaction is exothermicand the reaction vessel must be temperature controlled to maintainoptimal reaction kinetics. Specifically, the oxidation phase isextremely exothermic. Due to the strongly exothermic nature of thereaction, the hydrogen peroxide therefore can be added slowly so thatvolatile components, for example ethers, are not vaporized due to thereaction heat. In some embodiments, the reaction vessel may be cooled byinternal cooling coils. The cooling coils may be supplied with a coolantby means of an external gas/liquid refrigeration unit. In someembodiments, an internal temperature of the reaction vessel ismaintained at less than 10° C., 5° C., 1° C. or 0° C. In someembodiments, the reaction vessel is maintained at about −5° C. duringthe hydroboration-oxidation reaction.

In certain embodiments, the protected diol can have a percentcrystallinity of a salt, hydrate, solvate or crystalline form of diol tobe at least 100/o, at least 20/o, at least 30%, at least 40%, at least50%, at least, 60%, at least 70′, at least 80%, at least 90%, at least95%, or at least 99⁰%. In some embodiments, the percent crystallinitycan be substantially 100%, where substantially 100% indicates that theentire amount of diol appears to be crystalline as best can bedetermined using methods known in the art. Accordingly, therapeuticallyeffective amounts of diol can include amounts that vary incrystallinity. These include instances where an amount of thecrystallized diol in a solid form is subsequently dissolved, partiallydissolved, suspended or dispersed in a liquid.

Purification of OXY133

In some embodiments, the crude OXY133 must be separated from thereaction mixture prior to purification. In some embodiments, an organicsolvent such as dichloromethane is added to the crude OXY133 reactionmixture and the resulting organic layer is separated. Once separated,the crude OXY133 exists as a semi-solid viscous mass. The crude OXY133may be dissolved by any suitable means (e.g., dichloromethane, etc.) andplaced into a silica gel column with an organic solvent, such asmethanol-ethyl acetate, to solvate the crude OXY133. In someembodiments, the crude OXY133 may be crystallized or recrystallized. Insome embodiments, purified OXY133 is formed by recrystallizing the crudeOXY133 in a 3:1 mixture of acetone/water, as shown below:

As shown above, upon crystallization, the purified OXY133 forms ahydrate. However, it can be in the anhydrous form. In some embodiments,the percent crystallinity of any of the crystalline forms of OXY133described herein can vary with respect to the total amount of OXY133.

In certain embodiments, the OXY133 can have a percent crystallinity of asalt, hydrate, solvate or crystalline form of OXY133 to be at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least, 60%,at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.In some embodiments, the percent crystallinity can be substantially100%, where substantially 100% indicates that the entire amount ofOXY133 appears to be crystalline as best can be determined using methodsknown in the art. Accordingly, therapeutically effective amounts ofOXY133 can include amounts that vary in crystallinity. These includeinstances where an amount of the crystallized OXY133 in a solid form issubsequently dissolved, partially dissolved, or suspended or dispersedin a liquid.

In one embodiment, the purified OXY133 is crystallized as a monohydrate.However, in other embodiments the purified OXY133 may be crystallized inother hydrous forms, such as, for example, a dihydrate, a hemihydrate, asesquihydrate, a trihydrate, a tetrahydrate and the like, as well as thecorresponding solvated forms. In other embodiments, the purified OXY133is crystallized as a co-crystal or a pharmaceutically acceptable salt.

In some embodiments, the reaction mixture containing the crude OXY133may be solidified by mixing with heptanes. The product may subsequentlybe filtered and suspended in methylene chloride. In some embodiments,the crude OXY133 may be filtered from the suspension and crystallizedwith the use of acetone and water or other organic or inorganic solvents(e.g., diethyl ether, dichloromethane, ethyl acetate, acetone,n,n-dimethylformamide, acetonitrile, dimethyl sulfoxide, ammonia,t-butanol, n-propanol, ethanol, methanol, acetic acid or a combinationthereof).

In various embodiments, the crude OXY133 may be isolated and purified byany other traditional means. That is, the crude OXY133 can be isolatedand purified to the desired purity, e.g., from about 95% to about 99.9%by filtration, centrifugation, distillation to separate volatile liquidson the basis of their relative volatilities, crystallization,recrystallization, evaporation to remove volatile liquids fromnon-volatile solutes, solvent extraction to remove impurities,dissolving the composition in a solvent in which other components aresoluble therein or other purification methods. In various embodiments,the hydroboration-oxidation step and the purification step take place inthe same reaction vessel. In various embodiments, the alkylation step,the hydroboration-oxidation step and the purification step take place inthe same reaction vessel.

The method of synthesizing the intermediary diol (Formula 2) isstereoselective and produces a high yield of OXY133. For example, insome embodiments, the yield of the purified OXY133 is between about 20%and about 99%. In some embodiments, the yield of the purified OXY133 isbetween about 20% and about 80%. In some embodiments, the yield of thepurified OXY133 is between about 25% and about 70% or about 28%.However, it is contemplated that the percent yield may be higher orlower than these amounts.

In some embodiments, the purified OXY133 is formed in crystal form viacrystallization, which separates the OXY133 from the liquid feed streamby cooling the liquid feed stream or adding precipitants which lower thesolubility of byproducts and unused reactants in the reaction mixture sothat the OXY133 forms crystals. In some embodiments, the solid crystalsare then separated from the remaining liquor by filtration orcentrifugation. The crystals can be resolubilized in a solvent and thenrecrystallized and the crystals are then separated from the remainingliquor by filtration or centrifugation to obtain a highly pure sample ofOXY133. In some embodiments, the crystals can then be granulated to thedesired particle size.

In some embodiments, the purity of the OXY133 obtained is verifiedthrough nuclear magnetic resonance or mass spectroscopy. As shown inFIGS. 2-5, 1H NMR, 13C NMR, infrared spectroscopy, and mass spectroscopyanalysis indicated that the OXY133 product had high purity (e.g., having98% to about 99.99% by weight purity).

In some embodiments, the crude OXY133 can be purified where the purifiedOXY133 is formed in crystallized form in a solvent and then removed fromthe solvent to form a high purity OXY133 having a purity of from about98% to about 99.99%. In some embodiments, the OXY133 can be recoveredvia filtration or vacuum filtration before or after purification.

Methods of Making Pregnenolone Derivatives

The compound OXY133 can act as a potent osteogenic oxysterol, whichinduces osteogenic differentiation of osteoprogenitor cells in vitro androbust bone formation in vivo in rat and rabbit spine fusion models. Asa result, OXY133 is a drug candidate for local administration withpotential application in spine fusion and repair of non-union fractures.In various embodiments, OXY133 based therapeutic derivatives can be usedas bone specific drug delivery systems for osteogenic drugs or moleculesnot previously tested for systemic bone disease or chemical entitieswith high bone affinity, potentially rendering them into effectivetreatments for osteopenia or osteoporosis.

FIG. 8 is a schematic of a synthesis of oxysterol therapeuticderivatives or analogs according to one embodiment of this application.In particular, FIG. 8 illustrates methods of preparing C3 protectedpregnenolone derivatives, C3 protected diol derivatives and C3 protectedOXY133 derivatives as described below.

In some embodiments, this disclosure provides methods for thepreparation of pregnenolone derivatives which are useful startingmaterials in preparing diol derivatives, which in turn are suitable forthe preparation of OXY133 analogues as illustrated in FIG. 8. Usefulpregnenolone derivatives include compounds illustrated as Formula I inFIG. 8:

wherein R1 can be methyl, ethyl, silyl or a carbamate group. In someembodiments, pregnenolone derivatives include compounds where R₁ can bemethyl, ethyl, tertiary butyl dimethylsilyl (TBS). Generally, methods ofsynthesizing pregnenolone derivatives are known in the art and includethe Williamson ether synthesis and the Ullmann condensation.

In certain embodiments, following the Williamson synthesis, in order toprovide an alkyl based ether at C3, pregnenolone is reacted with analkyl halide such as R₁X in the presence of a strong base. In variousembodiments, R₁ can be C₁-C₂₀ primary alkyl group including, forexample, methyl, ethyl, propyl and X is a halide, for example, afluoride, chloride, bromide, iodide, or astatide. Useful bases includestrong bases, for example, sodium hydroxide, potassium hydroxide orcalcium hydroxide.

In other embodiments, useful pregnenolone derivatives include aromaticgroups at the C3 position, for example, (C₂-C₂₀) aryl or heteroarylgroups. In some embodiments, in order to couple a phenol group at the C3position of pregnenolone, the Ullman condensation or Williamson ethersynthesis can be used. In the Ullmann condensation, an aromatic alcoholsuch as pregnenolone can be reacted with an aromatic halide in thepresence of copper and a strong base at about 160° C. to yield anaromatic ether at C3 of pregnenolone. As in the Williamson ethersynthesis, useful strong bases include potassium hydroxide, sodiumhydroxide or calcium hydroxide.

Methods of Making Diol Derivatives

In certain embodiments, methods for the preparation of an intermediaryC3 protected diol useful in the production of OXY133 analogs orderivatives are provided.

In various embodiments, the method of making a derivative of anoxysterol comprises reacting a pregnenolone derivative of Formula I:

with an organometallic compound to form a C3 protected diol derivativeidentified as Formula II in FIG. 8:

wherein R₁ is methyl, ethyl, carbamate, or silyl group and R₃ is(C₆-C₂₆) alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno, heteroalkyleno, carbamate, benzyl or silyl group.

Subsequently, the diol derivative of Formula II can be subjected tohydroboration-oxidation to form a derivative of the oxysterol OXY133protected at C3 or a pharmaceutically acceptable salt thereof identifiedas Formula III in FIG. 8:

In some embodiments the organometallic compound useful to attach R₃ atC20 corresponds to the formula R₃MgX, where X is a halide and R₃comprises an aliphatic or cyclic substituent having at least one carbon.In other embodiments, the organometallic compound useful to form a C3protected diol derivative comprises or corresponds to the formula R₃Li,where R₃ comprises an aliphatic or cyclic substituent having at leastone carbon; and R₁ comprises methyl, ethyl or silyl. In someembodiments, R₃ is a hexyl group.

The method of synthesizing the intermediary C3 protected diol (FormulaII) is stereoselective and produces a high yield of the C3 protecteddiol. For example, in some embodiments, the yield of the desiredstereoisomer of the diol is between about 60% and about 70%. In someembodiments, the yield of the desired stereoisomer of the diol isbetween about 50% and about 60%. However, it is contemplated that thepercent yield may be higher or lower than these amounts. For example,the percent yield of Formula 2 as shown above may be about 200, 25%, 30,35%, 400, 45%, 50°, 55%, 60%, 65%, 70, 75%, 80%, 85%, 90% or 95%. Insome embodiments, the percent yield may be above 95%.

In various embodiments, the alkylation reaction with a Grignard reagentis carried out in a polar organic solvent, such as tetrahydrofuran.However, the reaction may be carried out in a variety of polar organicsolvents. For example, the reaction may be carried out in diethyl ether,ethyl ether, dimethyl ether or the like.

In some embodiments, the organometallic Grignard reagent comprisesn-hexylmagnesium chloride. However, in some embodiments, the alkylationreaction may be carried out with the use of an alkyllithium, such as,for example, n-hexyllithium. In various embodiments, the organometallicreagent includes an alkyl halide. For example, the organometallicreagent may have the following formula:

R₃—Mg—X,

where Mg comprises magnesium, X comprises chlorine, bromine, fluorine,iodine, or astatine and R₃ comprises an alkyl, a heteroalkyl, analkanyl, a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkynyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an aryleno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R₃ substituent comprises a (C₁-C₂₀)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroarylalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R₃ substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R₃ substituent is an aliphatic group. In someembodiments, the R₃ substituent is a cyclic group. In some embodiments,the R₃ substituent is a hexyl group.

Alternatively, the organometallic reagent may comprise the formula:

R₃—Li,

where Li comprises lithium and R₃ comprises an alkyl, a heteroalkyl, analkanyl, a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, aheteroalkanyl, an alkyldiyl, a heteroalkyldiyl, an alkyleno, aheteroalkyleno, an aryl, an aryldiyl, an aryleno, an arylaryl, a biaryl,an arylalkyl, a heteroaryl, a heteroaryldiyl, a heteroaryleno, aheteroaryl-heteroaryl, a biheteroaryl, a heteroarylalkyl or combinationsthereof. In some embodiments, the R₃ substituent comprises a (C₁-C₂₀)alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆)arylalkyl or heteroarylalkyl and a (C₅-C₂₀) arylalkyl orheteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a(C₄-C₁₀) alkyleno or heteroalkyleno. The R₃ substituent may be cyclic oracyclic, branched or unbranched, substituted or unsubstituted, aromatic,saturated or unsaturated chains, or combinations thereof. In someembodiments, the R₃ substituent is an aliphatic group. In someembodiments, the R₃ substituent is a cyclic group. In some embodiments,the R₃ substituent is a hexyl group.

In some embodiments, the alkylation reaction is exothermic and thereaction vessel may be temperature controlled to maintain optimalreaction kinetics. In some embodiments, the exothermic reaction releasesabout 1000 BTU per pound of solution. Due to the strongly exothermicnature of the reaction, the Grignard reagent therefore can be addedslowly so that volatile components, for example ethers, are notvaporized due to the reaction heat. In some embodiments, the reactionvessel may be cooled by internal cooling coils. The cooling coils may besupplied with a coolant by means of an external gas/liquid refrigerationunit. In some embodiments, an internal temperature of the reactionvessel is maintained at less than 15° C., 10° C., 5° C. or 1° C. In someembodiments, the reaction vessel is maintained at about 0° C. during thealkylation reaction to form the intermediary C3 protected diol ofFormula II.

In various embodiments, the C3 protected diol of Formula II issynthesized along with byproducts and can be purified. For example, theresulting C3 protected diol of Formula II may be a byproduct of adiastereomeric mixture. In various embodiments, the C3 protected diol ofFormula II may be isolated and purified. That is, the C3 protected diolof Formula II can be isolated and purified to the desired purity, e.g.,from about 95% to about 99.9% by filtration, centrifugation,distillation, which separates volatile liquids on the basis of theirrelative volatilities, crystallization, recrystallization, evaporationto remove volatile liquids from non-volatile solutes, solvent extractionto remove impurities, dissolving the composition in a solvent in whichother components are soluble therein or other purification methods. TheC3 protected diol may be purified by contacting it with organic and/orinorganic solvents, for example, THF, water, diethyl ether,dichloromethane, ethyl acetate, acetone, n,n-dimethylformamide,acetonitrile, dimethyl sulfoxide, ammonia, t-butanol, n-propanol,ethanol, methanol, acetic acid, or a combination thereof.

In various embodiments, the alkylation step and the purification steptake place in the same reaction vessel.

In some embodiments, the C3 protected diol is quenched with aqueousammonium chloride or acetic acid to reduce the amount of anions presentand to neutralize the reaction and separate it from the resultingorganic layer. The separated residue is recovered by evaporation andpurified by silica gel column chromatography.

The C3 protected diol may be anhydrous or in the monohydrate form.However, in other embodiments, the purified diol may be crystallized inother hydrous forms, such as, for example, a dihydrate, a hemihydrate, asesquihydrate, a trihydrate, a tetrahydrate or the like, as well as thecorresponding solvated forms. In other embodiments, the purifiedprotected diol is crystallized as a co-crystal or a pharmaceuticallyacceptable salt.

Methods of Making an Oxysterol-Therapeutic Agent Derivative

With further reference to FIG. 8, the method of synthesizing OXY133protected at C3 as disclosed herein includes reacting the C3 protecteddiol synthesized as described above with borane followed by oxidationwith hydrogen peroxide water in the reaction scheme shown below:

In some embodiments, crude and unpurified C3 protected OXY133 isproduced through a hydroboration and oxidation reaction of theintermediary protected diol having Formula II in reaction scheme 6.Borane compounds that can be used in the reaction include BH₃, B₂H₆.BH₃S(CH₃)₂ (BMS), borane adducts with phosphines and amines, e.g.,borane triethylamine; monosubstituted boranes of the form RBH₂ whereR=alkyl and halide, monoalkyl boranes (e.g., IpcBH2,monoisopinocampheylborane), monobromo- and monochloro-borane, complexesof monochloroborane and 1,4-dioxane, disubstituted boranes includingbulky boranes, such as for example, dialkylborane compounds such asdiethylborane, bis-3-methyl-2-butylborane (disiamylborane),9-borabycyclo[3,3,1]nonane (9-BBN), disiamylborane (Sia₂BH),dicyclohexylborane (Chx2BH), trialkylboranes, dialkylhalogenoboranes,dimesitylborane (C₆H₂Me₃)₂BH, alkenylboranes, pinacolborane, orcatecholborane or a combination thereof.

Briefly, as illustrated in scheme 6, a hydroboration and oxidationreaction is a two-step reaction. The boron and hydrogen add across thedouble bond of an alkene to form a complex with the alkene. Thus theboration phase of the reaction is stereoselective and regioselective.The oxidation phase of the reaction involves basic aqueous hydrogenperoxide to furnish a hydroxyl substituent in place of the boron. SeeVollhart, K P, Schore, N E, 2007, Organic Chemistry: Structure andFunction, Fifth Ed., New York, N.Y., Custom Publishing Company. Thus,the C3 protected intermediary diol having Formula III is reacted withborane and hydrogen peroxide to form crude OXY133 protected at C3. Insome embodiments, the step of forming crude C3 protected OXY133 takesplace in the same reaction vessel as the alkylation reaction. In otherembodiments, the step of forming crude C3 protected OXY133 takes placein a different reaction vessel as the alkylation reaction.

The hydroboration-oxidation step of the synthesis of C3 protectedOXY133, like the step of forming the intermediary diol, isstereoselective and produces a high yield. For example, in someembodiments, the percent yield of crude OXY133 may be higher or lowerthan these amounts. For example, the percent yield of Formula 2 as shownabove may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95%. In some embodiments, the percent yieldmay be above 95%.

In various embodiments, the hydroboration-oxidation reaction is carriedout in a polar organic solvent, such as tetrahydrofuran. However, thereaction may be carried out in a variety of polar organic solvents. Forexample, the reaction may be carried out in diethyl ether, ethyl ether,dimethyl ether or the like.

In some embodiments, the hydroboration-oxidation reaction is exothermicand the reaction vessel can be temperature controlled to maintainoptimal reaction kinetics. Specifically, the oxidation phase isextremely exothermic. Due to the strongly exothermic nature of thereaction, the hydrogen peroxide therefore can be added slowly so thatvolatile components, for example ethers, are not vaporized due to thereaction heat. In some embodiments, the reaction vessel may be cooled byinternal cooling coils. The cooling coils may be supplied with a coolantby means of an external gas/liquid refrigeration unit. In someembodiments, an internal temperature of the reaction vessel ismaintained at less than 10° C., 5° C., 1° C. or 0° C. In someembodiments, the reaction vessel is maintained at about −5° C. duringthe hydroboration-oxidation reaction.

In certain embodiments the C3 protected diol can have a percentcrystallinity of a salt, hydrate, solvate or crystalline form of diol tobe at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%. In some embodiments, the percent crystallinity can besubstantially 100%, where substantially 100% indicates that the entireamount of the C3 protected diol appears to be crystalline as best can bedetermined using methods known in the art. Accordingly, therapeuticallyeffective amounts of diol can include amounts that vary incrystallinity. These include instances where an amount of the C3protected crystallized diol in a solid form is subsequently dissolved,partially dissolved, or suspended or dispersed in a liquid.

When considering the C3 protected OXY133 molecule of Formula III, it isevident that there are only two other sites available for the formationof a derivative or an analogue with another compound moiety, namely atC6 and C20. In the C3 protected OXY133 molecule of Formula III, theC6-hydroxyl group is more reactive to another drug rather than thehydroxyl group at the C20 location because there are no other groupsaround it to impede reaction with the hydroxyl group as opposed to thehydroxyl group at the C20 position, which has a straight-chain alkylgroup. In other embodiments, it is possible to protect OXY133 moleculeat C6 and/or C20 thereby providing an analogue where the therapeuticagent is attached to C3 or C20 or at all active sites, C3, C6 and C20 ofthe OXY133 molecule.

In various embodiments, the C3 protected OXY133 molecule can reactthrough the C6-hydroxyl group with a therapeutic agent having known boneaffinity including bisphosphonates, antibiotics, proteins, moieties orfragments thereof. In FIG. 8, OXY133 substituted with a therapeuticagent is shown as formula IV:

wherein R1 and R3 are as described above and R4 is a therapeutic agentincluding bisphosphonates moieties, antibiotics moieties or proteinfragments.

Once formed, the C3 protected OXY133 therapeutic agent derivative can bedeprotected at C3 using classical chemistry methods known to one ofordinary skill in the art. For example, if the protecting group at C3 issilyl, in order to deprotect it, a fluoride source, such astetra-n-butylammonium fluoride (TBAF) or an HF pyridine complex can beused. In other aspects, when the protecting group at C3 is methyl oranother straight chain alkyl, an iodine source such as trimethylsilyliodide, (CH₃)₃SiI, iodotrimethylsilane (TMSI) can be used. Othersuitable deprotection methods can be used depending on the identity ofthe protecting group at C3. Upon deprotection, an OXY133 therapeuticderivative or analog is formed and is illustrated in FIG. 8 as FormulaIVb:

The entire synthesis of oxysterol analog or derivative is illustrated inFIG. 8. An embodiment of this application relates to an oxysterolderivative or analogue where the therapeutic agent is a bisphosphonate.Bisphosphonates are a class of drugs that are used to treat conditionsthat affect the bones. They inhibit mineralization or resorption of thebone by blocking the action of osteoclasts. Bisphosphonates areenzyme-resistant analogues of pyrophosphate, which normally inhibitsmineralization in the bone. Dose dependent, bisphosphonates reduce theturnover of bone by inhibiting recruitment and promoting apoptosis ofosteoclasts.

There are two classes of bisphosphonates, the nitrogenousbisphosphonates and the non-nitrogenous bisphosphonates. The nitrogenousbisphosphonates useful to prepare substituted oxysterol derivativesinclude without limitations alendronate, ibandronate, risedronate,zoledronate, olpadronate, neridronate or pamidronate. Thenon-nitrogenous bisphosphonates useful in this application includewithout limitations etidronate, clodronate or tiludronate. Thebisphosphonate will impart bone strengthening activity to the oxysteroland reduce or prevent bone loss useful in conditions, such as forexample, osteoporosis.

Another embodiment of this application relates to an oxysterolderivative where the therapeutic agent is an antibiotic, an antibioticmoiety or fragment or an antimicrobial agent or fragment thereof. Thiswill impart antimicrobial activity to the oxysterol and reduce orprevent infection. Some exemplary antibiotic or antimicrobial agentsinclude, by way of illustration and not limitation, acedapsone;acetosulfone sodium; alamecin; alexidine; amdinocillin; amdinocillinpivoxil; amicycline; amifloxacin; amifloxacin mesylate; amikacin;amikacin sulfate; aminoglycosides; aminosalicylic acid; aminosalicylatesodium; amoxicillin; amphomycin; ampicillin; ampicillin sodium;apalcillin sodium; apramycin; aspartocin; astromicin sulfate;avilamycin; avoparcin; azactam; azithromycin; azlocillin; azlocillinsodium; bacampicillin hydrochloride; bacitracin; bacitracin methylenedisalicylate; bacitracin zinc; bambermycins; benzoylpas calcium;berythromycin; betamicin sulfate; beta-lactamases; biapenem;biniramycin; biphenamine hydrochloride; bispyrithione magsulfex;butikacin; butirosin sulfate; capreomycin sulfate; carbadox;carbenicillin disodium; carbenicillin indanyl sodium; carbenicillinphenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor;cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium;cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium;cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol;cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium;cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide;cefotaxime sodium; cefotetan; cefotetan disodium; cefotiamhydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizolesodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoximeproxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime;ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime;cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrilesodium; cephalexin; cephalexin hydrochloride; cephaloglycin;cephaloridine; cephalothin sodium; cephapirin sodium; cephradine;cetocycline hydrochloride; cetophenicol; chloramphenicol;chloramphenicol palmitate; chloramphenicol pantothenate complex;chloramphenicol sodium succinate; chlorhexidine phosphanilate;chloroxylenol; chlortetracycline bisulfate; chlortetracyclinehydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride;cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin;clindamycin hydrochloride; clindamycin palmitate hydrochloride;clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillinsodium; chlorhexidine, cloxyquin; colistimethate sodium; colistinsulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine;dalfopristin; dapsone; daptomycin; demeclocycline; demeclocyclinehydrochloride; demecycline; denofungin; diaveridine; dicloxacillin;dicloxacillin sodium; dihydrostreptomycin sulfate; dipyrithione;dirithromycin; doxycycline; doxycycline calcium; doxycycline fosfatex;doxycycline hyclate; droxacin sodium; enoxacin; epicillin;epitetracycline hydrochloride; erythromycin; erythromycin acistrate;erythromycin estolate; erythromycin ethylsuccinate; erythromycingluceptate; erythromycin lactobionate; erythromycin propionate;erythromycin stearate; ethambutol hydrochloride; ethionamide;fleroxacin; floxacillin; fludalanine; flumequine; fluoroquinolones:fosfomycin; fosfomycin tromethamine; fumoxicillin; furazolium chloride;furazolium tartrate; fusidate sodium; fusidic acid; ganciclovir andganciclovir sodium; gentamicin sulfate; gloximonam; gramicidin;haloprogin; hetacillin; hetacillin potassium; hexedine; ibafloxacin;imipenem; isoconazole; isepamicin; isoniazid; josamycin; kanamycinsulfate; kitasamycin; levofuraltadone; levopropylcillin potassium;lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin;lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef;macrolides; mafenide; meclocycline; meclocycline sulfosalicylate;megalomicin potassium phosphate; mequidox; meropenem; methacycline;methacycline hydrochloride; methenamine; methenamine hippurate;methenamine mandelate; methicillin sodium; metioprim; metronidazolehydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium;minocycline; minocycline hydrochloride; mirincamycin hydrochloride;monensin; monensin sodiumr; nafcillin sodium; nalidixate sodium;nalidixic acid; natainycin; nebramycin; neomycin palmitate; neomycinsulfate; sulfonamide: neomycin undecylenate; netilmicin sulfate;neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone;nifurdazil: nifurimide; nifiupirinol: nifurquinazol; nifurthiazole;nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium;ofloxacin; onnetoprim; oxacillin and oxacillin sodium; oximonam;oximonam sodium; oxolinic acid; oxytetracycline; oxytetracyclinecalcium; oxytetracycline hydrochloride; paldimycin; parachlorophenol;paulomycin; pefloxacin; pefloxacin mesylate; penamecillin; penicillinssuch as penicillin g benzathine, penicillin g potassium, penicillin gprocaine, penicillin g sodium, penicillin v, penicillin v benzathine,penicillin v hydrabamine, and penicillin v potassium; pentizidonesodium; phenyl aminosalicylate; piperacillin sodium; pirbenicillinsodium; piridicillin sodium; pirlimycin hydrochloride; pivampicillinhydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxinB sulfate; porfiromycin; propikacin; pyrazinamide; pyrithione zinc;quindecamine acetate; quinupristin; racephenicol; ramoplanin; ranimycin;relomycin; repromicin; rifabutin; rifametane; rifamexil; rifamide;rifampin; rifapentine; rifaximin; rolitetracycline; rolitetracyclinenitrate; rosaramicin; rosaramicin butyrate; rosaramicin propionate;rosaramicin sodium phosphate; rosaramicin stearate; rosoxacin;roxarsone; roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin;spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride;steffimycin; streptomycin sulfate; streptonicozid; sulfabenz;sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine;sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene;sulfamerazine; sulfameter; sulfamethazine; sulfamethizole;sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc;sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet;sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine;sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillinhydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin;tetracycline; tetracycline hydrochloride; tetracycline phosphatecomplex; tetroxoprim; thiamphenicol; thiphencillin potassium;ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium;ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate;tosufloxacin; trimethoprim; trimethoprim sulfate;trimethoprim-sulfamthoxazole; trisulfapyrimidines; troleandomycin;trospectomycin sulfate; tyrothricin; vancomycin; vancomycinhydrochloride; virginiamycin; zorbamycin; or combinations thereof.

In yet other embodiments, the oxysterol therapeutic derivatives of thisapplication contain proteins as the therapeutic agent. Some exemplaryproteins or proteinaceous material include, by way of illustration andnot limitation, bone morphogenetic or morphogenic proteins (BMPs), boneinductive proteins, bone growth or growth factors, osteogenic proteins,or osteoinductive proteins. While these factors have different effectsand functions, as discussed herein, these will be referred tocollectively herein as osteoinductive factors.

Osteoinductive agents can be polypeptides or polynucleotidescompositions. Polynucleotide compositions of the osteoinductive agentsinclude, but are not limited to, isolated Bone Morphogenetic Protein(BMP), Vascular Endothelial Growth Factor (VEGF), Connective TissueGrowth Factor (CTGF), Osteoprotegerin, Growth Differentiation Factors(GDFs), Cartilage Derived Morphogenic Proteins (CDMPs), LIMMineralization Proteins (LMPs), Platelet derived growth factor, (PDGF orrhPDGF), Insulin-like growth factor (IGF) or Transforming Growth Factorbeta (TGF-beta) polypeptides.

Polypeptide compositions of the osteoinductive agents include, but arenot limited to, full length proteins, fragments or variants thereof.Variants of the isolated osteoinductive agents include, but are notlimited to, polypeptide variants that are designed to increase theduration of activity of the osteoinductive agent in vivo. Preferredembodiments of variant osteoinductive agents include, but are notlimited to, full length proteins or fragments thereof that areconjugated to polyethylene glycol (PEG) moieties to increase theirhalf-life in vivo (also known as pegylation). Methods of pegylatingpolypeptides are well known in the art (See, e.g., U.S. Pat. No.6,552,170 and European Pat. No. 0,401,384 as examples of methods ofgenerating pegylated polypeptides). In some embodiments, theosteoinductive agent(s) are provided as fusion proteins. In oneembodiment, the osteoinductive agent(s) are available as fusion proteinswith the Fc portion of human IgG. In another embodiment, theosteoinductive agent(s) are available as hetero- or homodimers ormultimers. Examples of some fusion proteins include, but are not limitedto, ligand fusions between mature osteoinductive polypeptides and the Fcportion of human Immunoglobulin G (IgG). Methods of making fusionproteins and constructs encoding the same are well known in the art.

In some embodiments, the oxysterol therapeutic derivatives include asthe therapeutic agent the osteoinductive agents, which are one or moremembers of the family of Bone Morphogenetic Proteins (“BMPs”). BMPs area class of proteins thought to have osteoinductive or growth-promotingactivities on endogenous bone tissue, or function as pro-collagenprecursors. Known members of the BMP family include, but are not limitedto, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18 as wellas polynucleotides or polypeptides thereof, as well as maturepolypeptides or polynucleotides encoding the same.

BMPs utilized as osteoinductive agents comprise one or more of BMP-1;BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11;BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as anycombination of one or more of these BMPs, including full length BMPs orfragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof. Part or all of the BMP protein can be attached to the oxysteroland impart further osteoinductive properties to the oxysterol.

In another embodiment, the therapeutic agents are osteoinductive agentswhich include osteoclastogenesis inhibitors to inhibit bone resorptionof the bone tissue surrounding the site of implantation by osteoclasts.Osteoclast and osteoclastogenesis inhibitors include, but are notlimited to, osteoprotegerin polynucleotides or polypeptides, as well asmature osteoprotegerin proteins, polypeptides or polynucleotidesencoding the same. Osteoprotegerin is a member of the TNF-receptorsuperfamily and is an osteoblast-secreted decoy receptor that functionsas a negative regulator of bone resorption. This protein specificallybinds to its ligand, osteoprotegerin ligand (TNFSF11/OPGL), both ofwhich are key extracellular regulators of osteoclast development.

In another embodiment, illustrated in FIG. 9, the method of synthesizingan intermediary diol derivative protected at the C3 position includesreacting a pregnenolone derivative, for example, a pregnenolone ether,ester or carbamate with an organometallic reagent to facilitatealkylation at the C20 position of pregnenolone as illustrated below:

wherein R₁ can be an aliphatic or cyclic substituent having at least onecarbon. In other embodiments, R₁ can be (C₁-C₂₀) alkyl or heteroalkyl, a(C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl or heteroalkyl and a(C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl orheteroalkyldiyl, or a (C₄-C₁₀) alkyleno or heteroalkyleno or carbamateor benzyl or silyl. In some embodiments, R₁ can be methyl, ethyl orsilyl group. In other embodiments, after the C3 protected pregnenoloneis reacted with hexyl MgCl there is a hexyl group at C20 resulting in adiol derivative having formula IIa. However, in some embodiments, whenthere is an n-hexyl group at the C20 position of the protected diol,then R₁ cannot be tert-butyl dimethyl silyl (TBS). The diol derivativeof formula IIa, which has a hexyl moiety at C20 has been tested forbiological activity. Unexpectedly, the diol derivative of formula IIaexhibited more biological activity than diol derivatives where the sidechain at C20 was a pentyl group or less. Without being bound by theory,it is possible that the enhanced biological activity of the diolderivative of formula IIa is due to the fact that the structure offormula IIa is similar to the structure of natural cholesterol which hasa hexyl side chain at C20. As before, once the C3 protected diolderivative is formed, it is subjected to hydroboration-oxidation toobtain a C3 protected OXY133 derivative of formula IIIa:

In various embodiments, the C3 protected OXY133 derivative of formulaIIIa can be reacted with a therapeutic agent comprising a bisphosphonateor bisphosphonate moiety, an antibiotic or antibiotic moiety, a proteinor a protein fragment to provide a C3 protected substituted oxysterolsof formula IVa:

Once formed, the C3 protected OXY133 therapeutic agent derivative can bedeprotected at C3 using classical chemistry methods known to one ofordinary skill in the art. For example, if the protecting group at C3 issilyl, in order to deprotect it, a fluoride source, such astetra-n-butylammonium fluoride (TBAF) or an HF pyridine complex can beused. In other aspects, when the protecting group at C3 is methyl oranother straight chain alkyl, an iodine source such as trimethylsilyliodide, (CH₃)₃SiI, iodotrimethylsilane (TMSI) can be used. In yet otheraspects, depending upon the protecting group at C3 other suitablemethods of deprotection can be used. Upon deprotection of the C3protected substituted oxysterol of formula IVa, an OXY133 therapeuticderivative or analog is formed and is illustrated in FIG. 9 as FormulaIVc:

In certain embodiments, bone specific delivery agents such asbisphosphonates or bisphosphonates moieties, antibiotics or antibioticsmoieties, proteins or fragments thereof can be attached to C3 protectedOXY133 molecules via hydrolysable linker bonds, L as illustrated in FIG.10. With further reference to FIG. 10, a C3 protected OXY133 derivativeof formula IIIa is synthesized as discussed above in connection with theembodiment illustrated in FIG. 9. Formula IIIa is reacted with acompound which can provide a linker L and then with a therapeutic agentR₄ selected from a bisphosphonate moiety, an antibiotic moiety, or aprotein fragment. Following deprotection, as illustrated in FIG. 10, acompound of formula VIIIa is obtained:

In other embodiments, linker attachments for these bone specificdelivery agents include, but are not limited to, succinate-basedlinkers, aspartate based linkers and/or carbamate-based linkers. In oneembodiment, also illustrated in FIG. 10, the linker is succinate basedand is provided by reaction with succinic anhydride in the presence of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) via an esterlinkage. A subsequent reaction with a therapeutic agent results in theattachment of R₄ to the linker of the C3 protected OXY133 therapeuticanalogue. Following a deprotection step a product of formula IXa isobtained as illustrated in FIG. 10:

wherein R₄ is a therapeutic agent selected from a bisphosphonate moiety,an antibiotic moiety or a protein fragment.

In various aspects, bisphosphonate moieties, antibiotic moieties orprotein fragments can be attached to drug molecules via hydrolysablelinker bonds. Non-hydrolysable bonds may be used in cases where the drugmolecule after conjugation to the bisphosphonate moiety, antibioticmoiety or protein fragment retains pharmacological activity. Estergroups can be used, as they populate a favorable stability rangerelative to more labile thioesters and more stable amides (L. Gil etal., Bioorg. Med. Chem. 1999, 7, 901-919). The in vivo stability ofester groups can be further fine-tuned by substitutions placed adjacentto the ester group (T. C. Bruice et al., Bioorganic Mechanisms, Vol. 1,W. A. Benjamin, New York, 1966, 1-258). In some embodiments, a C3protected OXY133-therapeutic agent derivative can be suitable forsystemic dosing (oral, ip, or iv) that entails selective deposition inbone tissue followed by enzymatic linker hydrolysis and release of theosteogenic agent, OXY133 and the bisphosphonates, antibiotics, proteinsor fragments thereof at controlled rates into the target tissue. Suchattachment of moiety R₄ of a bisphosphonate moiety, an antibiotic moietyor a protein fragment to the C6-position of a C3 protected OXY133 toform the complex or derivative of C3 protected OXY133-bisphosphonatemoiety, OXY133-antibiotic moiety or OXY133-protein fragment can beachieved by a straightforward coupling to succinic anhydride via esterlinkage, as depicted below:

wherein R₁ is as defined above and R₄ is a moiety of a bisphosphonatemoiety, an antibiotic moiety or a protein fragment.

Linkers, L, useful in other embodiments for conjugation with C3protected OXY133 include without limitations aspartate based linkers,succinate based linkers or urethane based linkers as illustrated below:

In another aspect illustrated in FIG. 11, a C3 protected OXY133derivative of formula IIIa is synthesized by hydroboration/oxidation asdiscussed above in connection with the embodiments illustrated in FIGS.9 and 10. In FIG. 11, the precursors of C3 protected OXY133 derivativeof formula IIIa are C3 protected diol derivative having a hexyl sidegroup at C20, which corresponds to formula IIa and a pregnenolone offormula Ib, which was subjected to treatment with an alkyl halide suchas R₁X in the presence of a base and organometallic Grignard reagentcorresponding to n-hexylmagnesium chloride. A succinate-based linker canbe attached at the C6 position of the C3 protected OXY133 by coupling tosuccinic anhydride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) via an ester linkage. EDCI is generally used as acarboxyl activating agent for the coupling of primary amines, such as atetracycline moiety of Formula V, to yield the amide bond. Thetetracycline fragment corresponding to Formula V:

can then be coupled to the succinate based linker of the C3 protectedOXY133 derivative. The protective group R1 can be removed by a standarddeprotection as described above in connection with the embodimentsillustrated in FIGS. 9 and 10 to obtain OXY149 which has a structurecorresponding to Formula VI:

FIG. 12 illustrates yet another embodiment of this application. In FIG.12, the OH group at C3 is protected with a methyl group as provided bythe starting material of pregnenolone methyl ether corresponding toformula Ia:

The remaining steps to form OXY149 methyl ether follow the same steps asdiscussed in connection with the synthesis of OXY149 illustrated in FIG.11. OXY149 methyl ether corresponds to formula VIa, where OH at C3 isprotected by the methyl group of the ether:

FIG. 13 is another embodiment of an oxysterol derivative synthesis,wherein the oxysterol is reacted with an alendronate of formula Vb:

The resulting intermediate is deprotected to yield the oxysterolderivative of formula VIc:

The synthesis illustrated in FIG. 13 is similar to the synthesis of theoxysterol derivative shown in FIG. 11, except that after the C3protected OXY133 of formula IIIa is reacted with succinic anhydride andEDCI, the resulting intermediate is reacted with an alendronate offormula Vb to yield, after deprotection, the oxysterol derivative offormula VIc.

FIG. 14 is yet another embodiment of an oxysterol derivative synthesis,wherein the oxysterol is attached to a protein of formula Vc (BMP-2) anddeprotected to yield the oxysterol derivative of formula VId:

The synthesis illustrated in FIG. 14 is similar to the synthesis of theoxysterol derivative shown in FIG. 11, except that after the C3protected OXY133 of formula IIa is reacted with succinic anhydride andEDCI, the resulting intermediate is reacted with the protein BMP-2 offormula Vc to yield, after deprotection, the oxysterol derivative offormula VId.

Use of Oxysterol-Therapeutic Agent Derivatives or Analogues

Oxysterols are derivatives of cholesterol that have been shown to have arange of activities including cell apoptosis and cholesterolhomeostasis. A group of osteoinductive oxysterols have been identifiedthat stimulate mesenchymal stem cell differentiation in vitro, inducebone formation and support spinal fusion in vivo. Oxysterols are thoughtto affect bone formation through activation of the hedgehog signalingpathway.

In use, OXY133 provides therapeutic treatment for bone conditions.OXY133 facilitates bone formation, osteoblastic differentiation,osteomorphogenesis and/or osteoproliferation. Treatment can beadministered to treat open fractures and fractures at high risk ofnon-union, and in subjects with spinal disorders. That is, OXY133 caninduce spinal fusion and may help treat degenerative disc disease orarthritis affecting the lumbar or cervical vertebrae.

Mesenchymal stem cells treated with OXY133 have been shown to haveincreased osteoblast differentiation. Thus, in some embodiments, OXY133may be implanted into a spinal site with mesenchymal stem cells toinduce bone growth through osteoblast differentiation. Periosteum tissueis one tissue type that is involved early during normal bone fracturerepair process and can recruit various cell types (e.g., mesenchymalstem cells) and bone growth factors necessary for bone fracture repair.Thus, in some embodiments, periosteum tissue is utilized as a source ofmesenchymal stem cells and/or growth factors in a demineralized bonecomposition.

Recombinantly produced versions of naturally occurring human proteins,such as rhBMP-2 and rhPDGF, have been studied for decades for theirability to induce or enhance new bone formation. While these proteinshave been effective in supporting bone healing, there are drawbacks withrespect to the complexity of manufacturing and the associated costs. Oneway to address these drawbacks has been to identify small molecules thatregulate parts of the bone signaling pathways to stimulate or enhancebone healing. Examples include the osteoinductive oxysterols andtherapeutic agents including bisphosphonates, antibiotics, proteins,moieties or fragments thereof.

In some embodiments, the OXY133-therapeutic agent derivative may beimplanted or injected directly into a surgical site in a patient. Insome embodiments, the OXY133-therapeutic agent derivative obtained fromthe methods delineated above is in the form of a depot. In variousembodiments, a plurality of depots (e.g., pellets) can be administeredto a surgical site. In some embodiments, a plurality of depots areprovided (e.g., in a kit) and administered to a surgical site andtriangulate and/or surround the site needed for bone growth. In variousembodiments, a plurality of depots comprise about 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 depots. In some embodiments, a plasticizer is used to lowerglass translation temperature in order to affect stability of thedevice.

In various embodiments, the depot comprises OXY133-therapeutic agentderivative and a biodegradable polymer in amorphous, crystalline orsemicrystalline form; where the crystalline form may include polymorphs,solvates or hydrates.

In some embodiments, OXY133-therapeutic agent derivative is administeredin a device that is solid or in semi-solid form. The solid or semi-solidform of the device may have a pre-dosed viscosity in the range of about1 to about 2000 centipoise (cps), 1 to about 200 cps, or 1 to about 100cps. After the solid or semi-solid device is administered to the targetsite, the viscosity of the semi-solid or solid depot will increase andthe semi-solid will have a modulus of elasticity in the range of about1×10² to about 6×10⁵ dynes/cm², or 2×10⁴ to about 5×10⁵ dynes/cm², or5×10⁴ to about 5×10⁵ dynes/cm².

In various embodiments, the semi-solid or solid depot may comprise apolymer having a molecular weight (MW), as shown by the inherentviscosity, from about 0.10 dL/g to about 1.2 dL/g or from about 0.20dL/g to about 0.50 dL/g. Other inherent viscosity ranges include but arenot limited to about 0.05 to about 0.15 dL/g, about 0.10 to about 0.20dL/g, about 0.15 to about 0.25 dL/g, about 0.20 to about 0.30 dL/g,about 0.25 to about 0.35 dL/g, about 0.30 to about 0.35 dL/g, about 0.35to about 0.45 dL/g, about 0.40 to about 0.45 dL/g, about 0.45 to about0.55 dL/g, about 0.50 to about 0.70 dL/g, about 0.55 to about 0.6 dL/g,about 0.60 to about 0.80 dL/g, about 0.70 to about 0.90 dL/g, about 0.80to about 1.00 dL/g, about 0.90 to about 1.10 dL/g, about 1.0 to about1.2 dL/g, about 1.1 to about 1.3 dL/g, about 1.2 to about 1.4 dL/g,about 1.3 to about 1.5 dL/g, about 1.4 to about 1.6 dL/g, about 1.5 toabout 1.7 dL/g, about 1.6 to about 1.8 dL/g, about 1.7 to about 1.9dL/g, or about 1.8 to about 2.1 dL/g.

In some embodiments, the depot may not be fully biodegradable. Forexample, the device may comprise polyurethane, polyurea,polyether(amide), PEBA, thermoplastic elastomeric olefin, copolyester,and styrenic thermoplastic elastomer, steel, aluminum, stainless steel,titanium, metal alloys with high non-ferrous metal content and a lowrelative proportion of iron, carbon device, glass device, plastics,ceramics, methacrylates, poly (N-isopropylacrylamide), PEO-PPO-PEO(pluronics) or combinations thereof. Typically, these types of matricesmay need to be removed after a certain amount of time.

In various embodiments, the depot (e.g., device) may comprise abioerodible, a bioabsorbable, and/or a biodegradable biopolymer that mayprovide immediate release, or sustained release of theOXY133-therapeutic agent derivative. Examples of suitable sustainedrelease biopolymers include but are not limited to poly (alpha-hydroxyacids), poly (lactide-co-glycolide) (PLGA), polylactide (PLA),polyglycolide (PG), polyethylene glycol (PEG) conjugates of poly(alpha-hydroxy acids), poly(orthoester)s (POE), poly(esteramide)s,polyaspirins, polyphosphagenes, starch, pre-gelatinized starch,hyaluronic acid, chitosans, gelatin, alginates, albumin, fibrin, vitaminE compounds, such as alpha tocopheryl acetate, d-alpha tocopherylsuccinate, D,L-lactide, or L-lactide-caprolactone, dextrans,vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBTcopolymer (polyactive), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA, PEG-PLG,PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymers, SAIB (sucroseacetate isobutyrate) or combinations thereof.

In some embodiments, the depot comprises at least one biodegradablepolymer, wherein the at least one biodegradable polymer comprises one ormore of poly(lactide-co-glycolide) (PLGA), polylactide (PLA),polyglycolide (PGA), D-lactide, D,L-lactide, L-lactide,D,L-lactide-co-ε-caprolactone, L-lactide-co-ε-caprolactone,D,L-lactide-co-glycolide-co-ϵ-caprolactone,poly(D,L-lactide-co-caprolactone), poly(L-lactide-co-caprolactone),poly(D-lactide-co-caprolactone), poly(D,L-lactide), poly(D-lactide),poly(L-lactide), poly(esteramide) or a combination thereof.

In some embodiments, the depot comprises at least one biodegradablematerial in a wt % of about 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%,78%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 55%, 50%, 45%, 35%,25%, 20%, 15%, 10%, or 5% based on the total weight of the depot and theremainder is active and/or inactive pharmaceutical ingredients.

Mannitol, trehalose, dextran, mPEG and/or PEG may be used as aplasticizer for the polymer. In some embodiments, the polymer and/orplasticizer may also be coated on the depot to provide the desiredrelease profile. In some embodiments, the coating thickness may be thin,for example, from about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 micronsto thicker coatings 60, 65, 70, 75, 80, 85, 90, 95, 100 microns to delayrelease of the OXY133-therapeutic agent derivative, sterol, or diol fromthe depot (e.g., device). In some embodiments, the range of the coatingon the depot ranges from about 5 microns to about 250 microns or 5microns to about 200 microns to delay release from the device.

The depot (e.g., device) can be different sizes, shapes andconfigurations. There are several factors that can be taken intoconsideration in determining the size, shape and configuration of thedepot. For example, both the size and shape may allow for ease inpositioning the depot at the target tissue site. In addition, the shapeand size of the system should be selected so as to minimize or preventthe depot from moving after implantation. In various embodiments, thedepot can be shaped like a rod or a flat surface such as a film or sheet(e.g., ribbon-like) or the like. Flexibility may be a consideration soas to facilitate placement of the device.

Radiographic markers can be included on the device to permit the user toposition the depot (e.g., device) accurately into the target site of thepatient. These radiographic markers will also permit the user to trackmovement and degradation of the depot (e.g., device) at the site overtime. In this embodiment, the user may accurately position the depot(e.g., device) in the site using any of the numerous diagnostic imagingprocedures. Such diagnostic imaging procedures include, for example,X-ray imaging or fluoroscopy. Examples of such radiographic markersinclude, but are not limited to, barium, phosphate, bismuth, iodine,tantalum, tungsten, and/or metal beads or particles. In variousembodiments, the radiographic marker could be a spherical shape or aring around the depot (e.g., device).

In some embodiments, the OXY133-therapeutic agent derivative can beadministered to the target site using a “cannula” or “needle” that canbe a part of a delivery device e.g., a syringe, a gun delivery device,or any medical device suitable for the application of OXY133, sterol, ordiol to a targeted organ or anatomic region. The cannula or needle ofthe device is designed to cause minimal physical and psychologicaltrauma to the patient.

In some embodiments, the depot can be sutured to a target tissue siteusing a suturing needle. The dimensions of the needle, among otherthings, will depend on the site for implantation. For example, the widthof the muscle planes in different surgical procedures can vary from 1-40cm. Thus, the needle, in various embodiments, can be designed for thesespecific areas.

The embodiments of the present disclosure are useful as pharmaceuticalcompositions prepared with a therapeutically effective amount of acompound of the disclosure, as defined herein, and a pharmaceuticallyacceptable carrier or diluent. The compounds of the disclosure can beformulated as pharmaceutical compositions and administered to a subjectin need of treatment, for example a mammal, such as a human patient, ina variety of forms adapted to the chosen route of administration, forexample, orally, nasally, intraperitoneally, or parenterally, byintravenous, intramuscular, topical or subcutaneous routes, or byinjection into tissue.

Thus, compounds of the present disclosure may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier, or by inhalation or insufflation. They may be enclosed in hardor soft shell gelatin capsules, may be compressed into tablets, or maybe incorporated directly with the food of the patient's diet. For oraltherapeutic administration, the compounds may be combined with one ormore excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. The compounds may be combined with an inert powdered carrierand inhaled by the subject or insufflated. Such compositions andpreparations should contain at least 0.1% of a compound of an embodimentof the present disclosure. The percentage of the compositions andpreparations may, of course, be varied and may be between about 2% toabout 60% of the weight of a given unit dosage form. The amount ofcompound in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the compounds may beincorporated into sustained-release preparations and devices. Forexample, the compounds may be incorporated into time release capsules,time release tablets, time release pills, and time release polymers ornanoparticles.

The compounds described in this disclosure may also be administeredintravenously or intraperitoneally or subcutaneously by infusion orinjection. Solutions of the compounds can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the compounds which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse of agents delaying absorption, for example, aluminum monostearateand gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsin the required amount in the appropriate solvent with a variety of theother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze drying techniques, which yield a powder of the activeingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

For topical administration, the compounds may be applied in pure form.However, it may be desirable to administer them to the skin ascompositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols or glycols orwater/alcohol/glycol blends, in which the compounds can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied via absorbent pads,impregnated bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds to the skin are known to the art; for example, seeJacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508), all of which are hereby incorporated by reference.

Useful dosages of the compounds described in this disclosure can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949, which is hereby incorporated by reference.

For example, the concentration of the compounds in a liquid composition,such as a lotion, can be from about 0.1 to about 25% by weight, or fromabout 0.5-10% by weight. The concentration in a semi-solid or solidcomposition such as a gel or a powder can be from about 0.1 to about 5%by weight, or from about 0.5 to about 2.5% by weight.

The amount of the compounds required for use in treatment will vary notonly with the particular salt selected but also with the route ofadministration, the nature of the condition being treated, the age andcondition of the patient, and will be ultimately at the discretion ofthe attendant physician or clinician.

Effective dosages and routes of administration of the OXY133 derivativesof the disclosure are conventional. The exact amount (effective dose) ofthe agent will vary from subject to subject, depending on, for example,the species, age, weight and general or clinical condition of thesubject, the severity or mechanism of any disorder being treated, theparticular agent or vehicle used, the method and scheduling ofadministration, and the like. A therapeutically effective dose can bedetermined empirically, by conventional procedures known to those ofskill in the art. See, e.g., The Pharmacological Basis of Therapeutics,Goodman and Gilman, eds., Macmillan Publishing Co., New York. Forexample, an effective dose can be estimated initially either in cellculture assays or in suitable animal models. The animal model may alsobe used to determine the appropriate concentration ranges and routes ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeutic dose canalso be selected by analogy to dosages for comparable therapeuticagents.

The particular mode of administration and the dosage regimen will beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose will be in the range of from about0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg ofbody weight per day, such as above about 0.1 mg per kilogram, or in arange of from about 1 to about 10 mg per kilogram body weight of therecipient per day. For example, a suitable dose may be about 1 mg/kg, 10mg/kg, or 50 mg/kg of body weight per day.

The compounds are conveniently administered in unit dosage form; forexample, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, orabout 100 mg of active ingredient per unit dosage form.

The OXY133-therapeutic agent derivative compounds described in thisdisclosure can be administered to achieve peak plasma concentrations of,for example, from about 0.5 to about 75 μM, from about 1 to 50 μM, fromabout 2 to about 30 μM, or from about 5 to about 25 μM. Exemplary plasmaconcentrations include at least or no more than 0.25, 0.5, 1, 5, 10, 25,50, 75, 100, or 200 μM. For example, plasma levels may be from about 1to 100 micromolar or from about 10 to about 25 micromolar. This may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of the compounds, optionally in saline, or orally administeredas a bolus containing about 1-100 mg of the compounds. Desirable bloodlevels may be maintained by continuous infusion to provide about0.00005-5 mg per kg body weight per hour, for example at least or nomore than 0.00005, 0.0005, 0.005, 0.05, 0.5, or 5 mg/kg/hr.Alternatively, such levels can be obtained by intermittent infusionscontaining about 0.0002-20 mg per kg body weight, for example, at leastor no more than 0.0002, 0.002, 0.02, 0.2, 2, 20, or 50 mg of thecompounds per kg of body weight.

The OXY133-therapeutic agent derivative compounds may conveniently bepresented in a single dose or as divided doses administered atappropriate intervals, for example, as one dose per day or as two,three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator.

An aspect of the disclosure is a bioactive or pharmaceutical compositioncomprising an OXY133-therapeutic agent derivative or OXY133 derivativesset forth herein or a pharmaceutically acceptable salt or solvatethereof and a pharmaceutically acceptable carrier. These bioactive orpharmaceutical compositions are sometimes referred to herein as“pharmaceutical compositions” or “bioactive compositions of thedisclosure.” Sometimes the phrase “administration of a compound” is usedherein in the context of administration of this OXY133-therapeutic agentderivative compound to a subject (e.g., contacting the subject with thecompound). It is to be understood that the OXY133-therapeutic agentderivative compound for such a use can generally be in the form of apharmaceutical composition or bioactive composition comprising theOXY133-therapeutic derivative compound.

Another aspect of the disclosure is a method for inducing (stimulating,enhancing) a Hedgehog (Hh) pathway mediated response, in a cell ortissue, e.g., in a subject, comprising contacting the cell or tissuewith an effective amount (e.g., a therapeutically effective amount) ofthe oxysterol-therapeutic agent analogue or derivative, wherein theHedgehog (Hh) pathway mediated response is the stimulation ofosteoblastic differentiation, osteomorphogenesis, and/orosteoproliferation. The Hh mediated response can be useful inregenerative medicine.

Another aspect of the disclosure is a method for treating a subjecthaving a bone disorder, osteopenia, osteoporosis, or a bone fracture,comprising administering to the subject an effective amount of abioactive composition or pharmaceutical composition comprising aOXY133-therapeutic agent derivatives. The subject can be administeredthe bioactive composition or pharmaceutical composition at atherapeutically effective dose in an effective dosage form at a selectedinterval to, e.g., increase bone mass, ameliorate symptoms ofosteoporosis, or reduce, eliminate, prevent or treat other conditionswhich would benefit from an increase in osteomorphogenesis and/orosteoproliferation. The subject can be administered the bioactivecomposition or pharmaceutical composition at a therapeutically effectivedose in an effective dosage form at a selected interval to amelioratethe symptoms of osteoporosis. In one embodiment, the subject is treatedto induce bone formation by harvesting mammalian mesenchymal stem cells(e.g., from the subject or from a suitable mammal, or from a tissue orcell bank), treating the mammalian mesenchymal cells with a compound toinduce osteoblastic differentiation of the cells, and administering thedifferentiated cells to the subject.

In any of the methods of the disclosure, the OXY133-therapeutic agentderivative or OXY133 analogue can be administered to a cell, tissue ororgan by local administration. For example, the OXY133-therapeutic agentderivative can be applied locally with a cream or the like, or it can beinjected or otherwise introduced directly into a cell, tissue or organ,or it can be introduced with a suitable medical device (e.g., animplant). Alternatively, the compound can be administered systemically,e.g., orally, intravenously (through IV), or via injection such asintraperitoneal (ip) injection or subcutaneous injection.

Another aspect of the disclosure is a kit for carrying out one or moreof the methods described herein. The kit can comprise an effectiveamount (e.g., a therapeutically effective amount) of a compound,optionally in a container.

Another aspect of the disclosure is an implant for use in the body of asubject (e.g., an animal such as a human) comprising a substrate havinga surface. The surface or insides of the implant comprises a bioactivecomposition or pharmaceutical composition comprising OXY133-therapeuticagent derivative in an amount sufficient to induce bone formation in thesurrounding bone tissue.

In addition to the compounds set forth herein, other embodiments of thedisclosure encompass any and all individual stereoisomers at any of thestereocenters shown in the formulas, including diastereomers, racemates,enantiomers, and other isomers of the compounds. In embodiments of thedisclosure, all polymorphs and solvates of the compound, such ashydrates and those formed with organic solvents, are included. Suchsolvates can be crystalline solids having a substantially fixed molarratio of solute and solvent. Suitable solvents will be known by those ofordinary skill in the art, e.g., water, ethanol or dimethylsulfoxide.Such isomers, polymorphs, and solvates may be prepared by methods knownin the art, such as by regiospecific and/or enantioselective synthesisand resolution.

The ability to prepare salts depends on the acidity or basicity of theoxysterol-therapeutic agent derivative. Suitable salts of the compoundinclude, but are not limited to, acid addition salts, such as those madewith hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric,nitric, phosphoric, acetic, propionic, glycolic, lactic pyruvic,malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic,carbonic cinnamic, mandelic, methanesulfonic, ethanesulfonic,hydroxyethanesulfonic, benezenesulfonic, p-toluene sulfonic,cyclohexanesulfamic, salicyclic, p-aminosalicylic, 2-phenoxybenzoic, and2-acetoxybenzoic acid; salts made with saccharin; alkali metal salts,such as sodium and potassium salts; alkaline earth metal salts, such ascalcium and magnesium salts; and salts formed with organic or inorganicligands, such as quaternary ammonium salts.

Additional suitable salts include, but are not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mutate, napsylate, nitrate, N-methylglucamine ammoniumsalt, oleate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide, and valerate salts of the compounds described in thisdisclosure. It is to be understood that references to compounds hereininclude pharmaceutically acceptable salts or solvates thereof.

In any of the methods, compositions or kits of the disclosure,particularly for use in treating a subject, a composition of thedisclosure may optionally be in combination with one or more othersuitable therapeutic agents. Any therapeutic agent that is suitable fortreatment of a particular condition can be used. Suitable agents ordrugs will be evident to one of ordinary skill in the art. For example,for the treatment of bone disorders, a conventional therapeutic drug canbe used in combination with a composition of the disclosure. Some suchagents include, e.g., parathyroid hormone, sodium fluoride, insulin-likegrowth factor I (ILGF-I), insulin-like growth factor II (ILGF-II),transforming growth factor beta (TGF-β), a cytochrome P450 inhibitor, anosteogenic prostanoid, BMP 2, BMP 4, BMP 7, BMP 14, and/orbisphosphonates or other inhibitors of bone resorption.

In some embodiments, a composition or compound of this disclosure can beformulated as a pharmaceutical composition, which comprises acomposition of this disclosure and a pharmaceutically acceptablecarrier. The carrier is naturally selected to minimize any degradationof the active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art. For adiscussion of pharmaceutically acceptable carriers and other componentsof pharmaceutical compositions, see, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Company, 1990. Some suitablepharmaceutical carriers will be evident to a skilled worker and include,e.g., water (including sterile and/or deionized water), suitable buffers(such as PBS), physiological saline, cell culture medium (such as DMEM),artificial cerebral spinal fluid, dimethylsulfoxide (DMSO), or the like.

One of ordinary skill in the art will appreciate that a particularformulation of the disclosure will depend, at least in part, upon theparticular agent or combination of agents that is employed and thechosen route of administration. Accordingly, there is a wide variationof suitable formulations of compositions of the present disclosure. Somerepresentative formulations are discussed below. Others will be evidentto one of ordinary skill in the art. A compound can be administeredlocally or directly to a cell, tissue or organ in need of treatment, orit can be administered systemically.

Formulations or compositions suitable for oral administration cancomprise of liquid solutions, such as an effective amount of compounddissolved in diluents, such as water, saline, or fruit juice; capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as solid, granules or freeze-dried cells; solutions orsuspensions in an aqueous liquid; and oil-in-water emulsions orwater-in-oil emulsions. Tablet forms can include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Suitable formulationsfor oral delivery can also be incorporated into synthetic and naturalpolymeric microspheres, or other means to protect the agents of thepresent disclosure from degradation within the gastrointestinal tract.

Formulations suitable for parenteral administration (e.g., intravenous)include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain anti-oxidants, buffers, bacteriostats, and solutesthat render the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials, and can bestored in a freeze-dried (e.g., lyophilized) condition requiring onlythe addition of the sterile liquid carrier, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

A compound, alone or in combination with other therapeutic agentsincluding a OXY133-therapeutic agent derivative, can be made intoaerosol formulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; mouthwashescomprising the active ingredient in a suitable liquid carrier; orcreams, emulsions, suspensions, solutions, gels, pastes, foams,lubricants, sprays, suppositories, or the like.

Other suitable formulations include, e.g., hydrogels and polymerssuitable for timed release of a compound, or nanoparticles for smalldose delivery of a compound.

A person of ordinary skill in the art will appreciate that a suitable orappropriate formulation can be selected, adapted or developed based uponthe particular application at hand. In addition, the pharmaceuticalcompositions of the present disclosure may be prepared foradministration by a variety of different routes, whether systemic, localor both. Such examples include, but are not limited to, administrationsperformed intraarticularly, intracranially, intradermally,intrahepatically, intramuscularly, intraocularly, intraperitoneally,intrathecally, intravenously, subcutaneously, transdermally, or directlyinto a bone region atherosclerotic site, such as by direct injection,introduction with a catheter or other medical devise, topicalapplication, direct application, and/or by implanting a device into inan artery or other appropriate tissue site.

A compound may be formulated to be contained within, or adapted torelease by a surgical or medical device or implant. In certain aspects,an implant may be coated or otherwise treated with a compound. Forexample, hydrogels, or other polymers, such as biocompatible and/orbiodegradable polymers, may be used to coat an implant with thecompositions of the present disclosure (e.g., the composition may beadapted for use with a medical device by using a hydrogel or otherpolymer). Polymers and copolymers for coating medical devices with anagent are well-known in the art. Examples of medical devices andimplants include, but are not limited to, sutures and prostheses such asprosthetic joints, and can be in the shape, e.g., of a pin, screw, plateor prosthetic joint.

In some embodiments of the disclosure, a compound can stimulate orinhibit a therapeutic response, as measured by any of a variety ofconventional assays, by about 1%, 5%, 10%, 20%, 30%, 400, 50% 1500,200%, or more than that of an untreated control sample. Intermediatevalues in these ranges are also included.

Dosages for a compound can be in unit dosage form, such as a tablet orcapsule. The term “unit dosage form,” as used herein, refers tophysically discrete units suitable as unitary dosages for animal (e.g.,human) subjects, each unit containing a predetermined quantity of anagent of the disclosure, alone or in combination with other therapeuticagents, calculated in an amount sufficient to produce the desired effectin association with a pharmaceutically acceptable diluent, carrier, orvehicle.

One of ordinary skill in the art can routinely determine the appropriatedose, schedule, and method of administration for the exact formulationof the composition being used, in order to achieve the desired effectiveamount or effective concentration of the agent in the individualpatient. One of ordinary skill in the art also can readily determine anduse an appropriate indicator of the “effective concentration” of thecompounds by a direct or indirect analysis of appropriate patientsamples (e.g., blood and/or tissues), in addition to analyzing theappropriate clinical symptoms of the disease, disorder, or condition.

The exact dose of a compound or composition thereof administered to ananimal, such as a human, in the context of the present disclosure willvary from subject to subject, depending on the species, age, weight, andgeneral condition of the subject, the severity or mechanism of anydisorder being treated, the particular agent or vehicle used, its modeof administration, other medications the patient is taking, and otherfactors normally considered by an attending physician, when determiningan individual regimen and dose level appropriate for a particularpatient, and the like. The dose used to achieve a desired concentrationin vivo will be determined by the potency of the form of the compound,the pharmacodynamics associated with the compound in the host, with orwithout additional agents, the severity of the disease state of infectedindividuals, as well as, in the case of systemic administration, thebody weight and age of the individual. The size of the dose may also bedetermined by the existence of any adverse side effects that mayaccompany the particular agent, or composition thereof, employed. It isgenerally desirable, whenever possible, to keep adverse side effects toa minimum.

For example, a dose can be administered in the range of from about 5 ng(nanograms) to about 1000 mg (milligrams), or from about 100 ng to about600 mg, or from about 1 mg to about 500 mg, or from about 20 mg to about400 mg. For example, the dose can be selected to achieve a dose to bodyweight ratio of from about 0.0001 mg/kg to about 1500 mg/kg, or fromabout 1 mg/kg to about 1000 mg/kg, or from about 5 mg/kg to about 150mg/kg, or from about 20 mg/kg to about 100 mg/kg. For example, a dosageunit can be in the range of from about 1 ng to about 5000 mg, or fromabout 5 ng to about 1000 mg, or from about 100 ng to about 600 mg, orfrom about 1 mg to about 500 mg, or from about 20 mg to about 400 mg, orfrom about 40 mg to about 200 mg of a compound or a compositioncomprising a compound. In one embodiment of the disclosure, amounts of acompound described in this disclosure (e.g., a few grams) areadministered locally, such as in a spine fusion procedure as part of ascaffold.

A dose of the oxysterol-therapeutic agent derivative can be administeredonce per day, twice per day, four times per day, or more than four timesper day as required to elicit a desired therapeutic effect. For example,a dose administration regimen can be selected to achieve a blood serumconcentration of a compound of the present disclosure in the range offrom about 0.01 to about 1000 nM, or from about 0.1 to about 750 nM, orfrom about 1 to about 500 nM, or from about 20 to about 500 nM, or fromabout 100 to about 500 nM, or from about 200 to about 400 nM. Forexample, a dose administration regime can be selected to achieve anaverage blood serum concentration with a half maximum dose of a compoundof the present disclosure in the range of from about 1 μg/L (microgramper liter) to about 2000 μg/L, or from about 2 μg/L to about 1000 μg/L,or from about 5 μg/L to about 500 μg/L, or from about 10 μg/L to about400 μg/L, or from about 20 μg/L to about 200 μg/L, or from about 40 μg/Lto about 100 μg/L.

Certain embodiments of the disclosure may also include treatment with anadditional agent which acts independently or synergistically with theoxysterol-therapeutic agent derivative to improve the therapeuticresults. When given in combined therapy, the agent other than theoxysterol-therapeutic agent derivative can be given at the same time asthe compound, or the dosing can be staggered as desired. The two (ormore) drugs also can be combined in a composition. Doses of each can beless when used in combination than when either is used alone. Suitabledoses can be determined by a skilled worker, using standard dosageparameters.

In one embodiment of the disclosure, a kit is useful for any of themethods disclosed herein, either in vitro or in vivo. Such a kitcomprises a compound or a bioactive or pharmaceutical compositionthereof, and can comprise one or more other oxysterols, e.g., whichresult in an increase in an Hh pathway-mediated activity, or othersuitable therapeutic agents. Optionally, the kits comprise instructionsfor performing the method. Optional elements of a kit of the disclosureinclude suitable buffers, pharmaceutically acceptable carriers, or thelike, containers, or packaging materials. The reagents of the kit may bein containers in which the reagents are stable, e.g., in lyophilizedform or stabilized liquids. The reagents may also be in single use form,e.g., in single dosage form. One of ordinary skill in the art willrecognize components of kits suitable for carrying out any of themethods of the disclosure.

A variety of conditions can be treated with an oxysterol-therapeuticagent derivative, used alone or in combination with other therapeuticagents. An oxysterol-therapeutic agent derivative can result in anincrease in Hedgehog pathway activity.

One effect of an oxysterol-therapeutic agent derivative can be to targetpluripotent cells to induce their lineage specific differentiation intovarious cell types, e.g., osteoblasts. For example, mesenchymal stemcells treated with a compound can show induced expression of markers ofosteoblast differentiation. Without wishing to be bound by anyparticular mechanism, it is suggested that this lineage specificdifferentiation is due to the induction of Hedgehog signaling in thesecells. However, methods of treatment discussed herein are included inthe present disclosure, regardless of the mechanism by which thecompound functions. An oxysterol-therapeutic agent derivative can beuseful for treating conditions which would benefit from stimulation ofbone formation, osteoblastic differentiation, osteomorphogenesis and/orosteoproliferation. Among these conditions or treatments are, e.g.,osteoinductive therapy for stimulation of localized bone formation inspine fusion or osteoporosis, bone fracture repair or healing, dentalprocedures for which increased bone formation in the jaw is of clinicalbenefit, repair of craniofacial bone defects induced by trauma orcongenital defects such as cleft palate/lip, and a number of othermusculoskeletal disorders in which native bone growth is inadequate,which will be evident to skilled workers. Treatment can be administeredto treat open fractures and fractures at high risk of non-union, and insubjects with spinal disorders, including subjects in need of spinefusion (e.g., anterior lumbar interbody fusion, posterior lumbar spinalfusion, and cervical spine fusion) or subjects having degenerative discdisease or arthritis affecting the lumbar and cervical spine.Furthermore, an oxysterol-therapeutic agent derivative can be used totreat osteoporosis, particularly in the aging and post-menopausalpopulation, resulting from increased bone resorption by osteoclasts inparallel with decreased bone formation by osteoblasts.

More particularly, the following types of bone-related treatments can becarried out. In some embodiments, a compound can be used as anosteogenic agent delivered locally in the body in order to stimulatelocalized bone formation, using a scaffold that is composed of acompatible molecule such as but not limited to collagen I, which absorbsthe compound and then is placed inside the body. For example, thescaffold containing the oxysterol-therapeutic agent derivative can beplaced in between transverse processes or in the intervertebral discwhere the fusion of two or more vertebrae is indicated, for example inspinal fusion, pseudoarthrosis, and non-union fusions. In otherembodiments, the scaffold containing the oxysterol-therapeutic agentderivative is placed in a fractured bone in order to stimulate boneformation and healing of the fracture; is placed in a bone defect suchas calvarial or maxillofacial bone defects where bone regeneration bythe compound is indicated; or is placed in the jaw bone in order tostimulate bone formation as a means of regenerating bone prior to dentalprocedures such as dental implants. In other embodiments, anoxysterol-therapeutic agent derivative can be used as an osteogenicagent in vitro. For example, it can be administered to osteoprogenitorcells, for example mesenchymal stem cells, in order to stimulate theirosteogenic differentiation prior to the application of such cells inorthopedic and other procedures as indicated above in order to stimulatelocalized bone formation. In yet other embodiments, anoxysterol-therapeutic agent derivative can be used in vitro in order tostimulate the Hedgehog signaling pathway in osteoprogenitor cells,thereby leading to the osteogenic differentiation of the cells in vitroor in vivo.

In the foregoing and in the following examples, all temperatures are setforth in Celsius degrees; and, unless otherwise indicated, all parts andpercentages are by weight. The osteogenic oxysterols described above areuseful for direct, localized administration to target cells, tissues, ororgans of interest.

These and other aspects of the present application will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the applicationbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1

Preparation from Pregnenolone Acetate

8.25 mL n-hexylmagnesium chloride (2 M, 16.5 mmol) in THF was added to asolution of pregnenolone acetate in THF under vigorous electromagneticstirring and ice bath cooling. The pregnenolone acetate solutioncontained 1.79 g of compound 1, pregnenolone acetate, (5 mmol) in 4.5 mLTHF. The addition took place over 2 minutes. After addition wascompleted, the mixture was stirred at room temperature for 3.5 hours, atwhich point the mixture had turned to a gel. The gel was then digestedwith a mixture of saturated aqueous NH₄Cl and MTBE (methyltertiary-butyl ether). The organic layer was separated, washed withwater three times and evaporated. The residue was separated by silicagel column chromatography using an EtOAc (ethyl acetate)/petroleum ethermixture (ratio 70/30) to give compound 2, a diol, as a white solid. 1.29g (3.21 mmol) of the solid diol was extracted for a 64% isolated yield.The reaction is shown below in A:

The ¹H NMR data of the diol in CDCl₃ at 400 MHz illustrated thefollowing: δ: 0.8-1.9 (40H), 1.98 (m, 1H), 2.09 (m, 1H), 2.23 (m, 1H),2.29 (m, 1H), 3.52 (m, 1H), 5.35 (m, 1H), as shown in FIG. 6. The ¹³CNMR data of the diol in CDCl₃ at 100 MHz in FIG. 7 illustrated thefollowing: d: 13.6, 14.1, 19.4, 20.9, 22.4, 22.6, 23.8, 24.2, 26.4,30.0, 31.3, 31.6, 31.8, 31.9, 36.5, 37.3, 40.1, 42.3, 42.6, 44.0, 50.1,56.9, 57.6, 71.7, 75.2, 121.6, 140.8.

The diol created has an IUPAC nameof(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.

Example 2

Preparation from Pregnenolone

Alternatively to Example 1, compound 2 of reaction scheme A above can beprepared from pregnenolone shown below in scheme B utilizing the sameprocedure as utilized for the conversion of compound 1 to compound 2. Inthis procedure 10 g of pregnenolone was converted to 7.05 g of compound2, which accounted for a 55% yield.

2500 mL of n-hexylmagnesium chloride (2 M, 5 mol) was charged to areactor and the solution was cooled to −5° C. A solution of pregnenoloneacetate in THF was charged to the reactor at a rate which maintained theinternal reaction temperature below 1° C. The pregnenolone solutioncontained 500 g pregnenolone (1.4 mol) in 8 liters of THF. After theaddition was complete, the mixture was held at 0° C. for 1 hour thenallowed to warm to room temperature overnight. The reaction mixture hadbecome a solid, gelatinous mass. 2 liters of additional THF was addedfollowed by 10 ml of glacial acetic acid. The reaction mixture wascooled to 5° C. and quenched by the addition of 350 ml of glacial aceticacid which gave a solution. The reaction mixture was concentrated underreduced pressure to a thick syrup. The compound was dissolved indichloromethane, washed with water and finally washed with saturatedsodium bicarbonate. The organic layer was concentrated under reducedpressure to an amber oil. Mass recovery was about 800 grams. The crudematerial was utilized as-is in the next step.

The diol created has an IUPAC name of(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(S)-2-hydroxyoctan-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.

Example 3

The crude hexyl diol product (800 grams) was dissolved in 8 liters ofTHF, charged to a reactor, and was cooled to −5° C. 6300 mL ofborane-THF complex (1 M, 6.3 moles, 4.5 equivalents) in THF was chargedat a rate which maintained the internal reaction temperature below 1° C.Once the addition was complete, the reaction mixture was stirred at 0°C. for 1.5 hours then allowed to warm to room temperature overnight. Thereaction is shown below.

The reaction mixture was quenched by addition of a mixture of 10% sodiumhydroxide (4750 mL) and 30% hydrogen peroxide (1375 mL). The quench wasextremely exothermic and required several hours to complete. Theinternal temperature was maintained below 10° C. After the addition ofthe quench volume was complete, the mixture was held cold for 1.5 hoursthen allowed to warm to room temperature overnight. 8 liters ofdichloromethane was then added. The organic layer was isolated andwashed with 7 liters of fresh water, and was concentrated under reducedpressure. The product was isolated as a viscous, oily mass whichsolidified upon standing.

The product was dissolved in 4 liters of dichloromethane and was placedonto a silica gel column prepared in dichloromethane. The column waseluted first with 25% ethyl acetate to elute the 7-methyl-7-tridecylalcohol by-product. Subsequently, the column was eluted with 10%methanol-ethyl acetate to solvate the OXY133. The collected fractionswere combined and concentrated under reduced pressure to a waxy solid.The compound was dissolved in acetone-water mixture (3:1) andconcentrated under reduced pressure to remove residual solvents. Theresulting crude OXY133 was utilized in the next step.

Alternatively, the viscous product recovered from thehydroboration/oxidation can be solidified by stirring with heptanes, andthe product isolated by filtration. The isolated product is suspended inmethylene chloride (7.3 mL methylene chloride/g solid). The product wasisolated by filtration and used as-is in the next step.

Example 4

OXY133 was recrystallized by dissolving 630 grams of crude OXY133 into1500 ml of a 3:1 acetone/water mixture at reflux, then cooling to roomtemperature. The crystalline solid was recovered by vacuum filtrationand dried to afford 336 g, which was a 28% overall yield fromcompound 1. The OXY133 produced was monohydrous, and has an IUPAC nameof (3S,5S,6S,8R,9S,1OR,13S,14S,17S)-17-((S)-2-hydroxyoctan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[α]phenanthrene-3,6-diol,monohydrate.

FIG. 1 illustrates the step-wise reaction for synthesizing OXY133 withstarting reactants comprising pregnenolone acetate. The pregnenolone isreacted with an organometallic compound to produce a sterol or diolhaving two hydroxyl groups. The sterol or diol is then reacted withborane and hydrogen peroxide and purified to produce OXY133.

The ¹H NMR data of OXY133 in CDCl₃ at 400 MHz illustrated the following:δ: 0.66 (m, 1H), 0.85 (m, 10H), 1.23 (m, 18H), 1.47 (m, 9H), 1.68 (m,4H), 1.81 (m, 1H), 1.99 (m, 1H), 2.06 (m, 1H), 2.18 (m, 1H), 3.42 (m,1H), 3.58 (m, 1H). The ¹³C NMR data of OXY133 in CDCl₃ at 400 MHzillustrated the following: d: 13.7, 14.0, 14.3, 21.2, 22.5, 22.8, 23.9,24.4, 26.6, 30.1, 31.1, 32.1, 32.5, 33.9, 36.5, 37.5, 40.4, 41.7, 43.1,44.3, 51.9, 53.9, 56.5, 57.9, 69.6, 71.3, 75.4. The infraredspectroscopy data of OXY133 showed peaks at 3342 cm⁻¹, 2929 cm⁻¹, 2872cm⁻¹, 2849 cm⁻¹. The turbo spray mass spectrometry data of the OXY133showed peaks at 438.4 m/z [M+NH₄]+, 420.4 m/z (M-H₂O+NH₄]+, 403.4 m/z[M-H₂O+H]+, 385.4 m/z [M−2H₂O+H]+. The ¹H NMR, ¹³C NMR, IR, and MS ofOXY133 data are shown in FIGS. 2, 3, 4 and 5, respectively. FIG. 6 is agraphic illustration of ¹H NMR data obtained from the intermediarysterol or diol to synthesize OXY133. FIG. 7 is a graphic illustration of¹³C NMR data obtained from the intermediary sterol or diol to synthesizeOXY133;

Example 5

Alternative One-Vessel Procedure from Pregnenolone Acetate

100 mL n-hexylmagnesium chloride (2M in THF, 200 mmol) was charged to aflask and cooled to −10° C. A solution containing 20 g pregnenoloneacetate (56 mmol) in 200 ml of anhydrous THF was added dropwise, whilemaintaining the internal reaction temperature below −10° C. After theaddition was completed, the mixture was stirred for 30 minutes thenallowed to warm to room temperature. After 4 hours at room temperature,the mixture had become a gelatinous stirrable mass. The mixture wascooled to 0° C. and 200 mL Borane-THF complex (1M in THF, 200 mmol) wasadded dropwise, while maintaining the internal temperature below 0° C.Once addition was complete, the resulting solution was allowed to warmto room temperature overnight.

The mixture was cooled to 0° C. and quenched by the slow addition of amixture of 10% NaOH (190 mL) and 30% H₂O₂ (55 mL). Once the quench wascomplete, the mixture was extracted with MTBE (800 mL total) resultingin an emulsion. Brine was added and the layers were separated. Theorganic phase was concentrated under reduced pressure to a clear,viscous oil. The oil was further purified utilizing the plug columnmethod previously described.

It will be apparent to those of ordinary skill in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

What is claimed is:
 1. A method of making a derivative of an oxysterol, the method comprising (i) reacting a pregnenolone derivative of formula I:

with an organometallic compound to form a diol derivative of formula II:

(ii) reacting the diol derivative of formula II with a borane compound to form an oxysterol or a pharmaceutically acceptable salt thereof of formula III:

(iii) reacting the compound of formula III with a therapeutic agent or a fragment of a therapeutic agent to form an oxysterol derivative of formula IV:

wherein R₁ is a protecting group, R₃ is an aliphatic or cyclic substituent having at least one carbon and R₄ is a bisphosphonate moiety, an antibiotic moiety, a protein or a protein fragment.
 2. The method of claim 1, wherein the organometallic compound corresponds to the formula R₃MgX or R₃Li where X is a halide and R₃ is an aliphatic or cyclic substituent having at least one carbon.
 3. The method of claim 1, further comprising optionally deprotecting the compound of formula IV of R₁ to obtain a compound of formula IVb:

by treatment with an iodine or a fluoride source.
 4. The method of claim 1, wherein R₁ is methyl, ethyl, silyl or carbamate group and R₃ is (C₆-C₂₆) alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl or heteroalkyl or a (C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, a (C₄-C₁₀) alkyleno or heteroalkyleno or carbamate.
 5. The method of claim 1, wherein the pregnenolone derivative of formula II is prepared by reacting pregnenolone with R₁X in a base, wherein R₁ is methyl, ethyl, silyl or carbamate, X is an halide, wherein the base is NaOH, KOH or Ca(OH)₂.
 6. The method of claim 1, wherein R₃MgX is n-hexyl magnesium chloride and is reacted in tetrahydrofuran to obtain a diol derivative of the formula IIa:


7. The method of claim 6, wherein the compound of formula IIa is reacted with BH₃ to form a borane intermediate, the borane intermediate is reacted with hydrogen peroxide to form an OXY133 derivative of formula IIIa:


8. The method of claim 7, further comprising (i) reacting the compound of formula IIIa with a therapeutic agent R₄, wherein R₄ is a bisphosphonate moiety, an antibiotic moiety or a protein fragment to form a compound of formula IVa:

(ii) optionally, deprotecting the compound of formula IVa to obtain a compound of formula IVc:


9. The method of claim 6, wherein the pregnenolone derivative of formula IIa is prepared by reacting pregnenolone with R₁X in a base, wherein R₁ is methyl, ethyl, silyl or carbamate, X is a halide, and the base is NaOH, KOH or Ca(OH)₂.
 10. The method of claim 7, further comprising reacting the compound of formula IIIa with a compound comprising a linker L, the linker L comprising aspartate based linkers, succinate based linkers or urethane based linkers; coupling with a therapeutic agent R₄ to obtain a compound of formula VIII:

wherein R₄ is a bisphosphonate moiety, an antibiotic moiety, or a protein fragment; optionally, deprotecting the compound of formula VIII with an iodine source or a fluoride source to obtain a compound of formula VIIIa:


11. The method of claim 10, wherein when L is a succinate based linker and the oxysterol derivative is a compound of the formula IXa:


12. The method of claim 10, wherein the iodine source is trimethylsilyl iodide or the fluoride source is tetra-n-butylammonium fluoride or HF pyridine complex.
 13. The method of claim 7, further comprising reacting the compound of formula IIIa with a succinate based linker; and coupling with a compound of formula V:

to obtain a compound of formula VIb:


14. The method of claim 13, further comprising deprotecting the compound of formula VIb of R₁ by treatment with an iodine source or a fluoride source to obtain a compound of formula VI (OXY 149):


15. The method of claim 14, wherein the iodine source is trimethylsilyl iodide, the fluoride source is tetra-n-butylammonium fluoride or HF pyridine complex.
 16. A method of making OXY149 methyl ether, the method comprising forming pregnenolone methyl ether of formula Ia:

by reacting pregnenolone with CH₃X in a base, wherein X is a halide and the base is NaOH, KOH or Ca(OH)₂; reacting pregnenolone methyl ether of formula Ia with hexyl magnesium chloride in tetrahydrofuran to form pregnenolone diol methyl ether of formula IIb:

reacting pregnenolone diol methyl ether of formula IIb with BH₃ and reacting the resulting borane intermediate with hydrogen peroxide to form a OXY133 methyl ether or a pharmaceutically acceptable salt thereof of formula IIIb:

reacting the OXY133 methyl ether of formula IIIb with succinic anhydride to provide a succinate based linker and further reacting with a compound of formula V

to obtain a compound of formula VIa (OXY149 methyl ether)


17. The method of claim 1, wherein R₄ is a kanamycin moiety.
 18. The method of claim 9, wherein R₄ is a kanamycin moiety.
 19. The method of claim 17, wherein all reactions are carried out in a single container.
 20. A compound corresponding to the structure of formula X:

wherein R₁ is methyl, ethyl, silyl or carbamate group and R₃ is (C₆-C₂₆) alkyl or heteroalkyl, a (C₂-C₂₀) aryl or heteroaryl, a (C₆-C₂₆) arylalkyl or heteroalkyl and a (C₅-C₂₀) arylalkyl or heteroaryl-heteroalkyl, a (C₄-C₁₀) alkyldiyl or heteroalkyldiyl, or a (C₄-C₁₀) alkyleno or heteroalkyleno, carbamate, and L is a linker moiety, the linker moiety comprising an aspartate based linker, a succinate based linker or a urethane based linker and R₄ is a derivative of a therapeutic agent, the therapeutic agent comprising a bisphosphonate moiety, an antibiotic moiety, a protein or a protein fragment. 